T-140 peptide analogs having cxcr4 super-agonist activity for cancer therapy

ABSTRACT

The present invention is directed to novel therapeutic uses of T-140 analog peptides and compositions comprising same. Specifically, the invention provides compositions and methods useful in cancer therapy.

RELATED APPLICATION DATA

This application is divisional of U.S. patent application Ser. No.12/520,803 filed on Oct. 26, 2009, which is a National Phase of PCTPatent Application No. PCT/IL2007/001597 filed on Dec. 23, 2007, whichclaims the benefit of priority of U.S. Provisional Patent ApplicationNo. 60/876,145 filed on Dec. 21, 2006. The contents of all of the aboveapplications are incorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention is directed to compositions comprising T-140peptide analogs having CXCR4 super-agonist activity and to noveltherapeutic uses thereof in cancer therapy.

BACKGROUND OF THE INVENTION

Chemokines, a family of small pro-inflammatory cytokines, and theirreceptors, regulate a variety of immune responses to infection,inflammation and tissue repair. Chemokines are divided between two majorfamilies on the basis of relative position of cysteine residues in themature protein (C—C and C—X—C). Primarily, they are responsible for thedirectional migration, or chemotaxis, of lymphocytes to specificlymphoid tissues, and the recruitment of leukocytes to the sites ofinfection or tissue damage. In addition to their chemotactic function,chemokines are implicated in other biological events includingembryogenesis, lymphopoiesis, vascularization, and HIV pathogenesis.More recently, it has been established that cancer cells exploitsignaling through chemokine receptors for several key steps involved ininitiation and progression of primary and metastatic cancer. Differenttypes of cancers express different CC and CXC chemokine receptors. Thereis one chemokine receptor, however, that appears to be expressed by themajority of cancer types, namely, CXCR4.

The CXCR4/CXCL12 Axis

The chemokine receptor CXCR4 is a G-protein coupled receptor that isexpressed in a wide assortment of normal tissues, and plays afundamental role in fetal development, mobilization of hematopoieticstem cells and trafficking of naive lymphocytes (Rossi and Zlotnik,2000). Besides normal tissues, CXCR4 appears to be expressed by at least23 different epithelial, mesenchymal and hematopoietic cancers,including prostate cancer, and acute and chronic myeloid leukemias(Balkwill, 2004). The chemokine CXCL12 (also known as stromal-derivedfactor-1, or SDF-1) is CXCR4's only natural ligand. CXCL12 is expressedconstitutively in a variety of tissues, including lung, liver, bonemarrow and lymph nodes. These organs with highest expression of CXCL12correlate with common metastatic destinations in many cancers. Thechemokine receptor, CXCR4, and its ligand, CXCL12, appear to be animportant chemokine axis regulating tumor growth and metastasis(Nagasawa et al., 1994; Muller et al., 2001; Phillips et al., 2003).

Binding of CXCL12 to CXCR4 activates a variety of intracellular signaltransduction pathways and effector molecules that regulate cellchemotaxis, adhesion, survival, and proliferation. There are a number ofkey molecules that mediate signaling through CXCR4, and some of themwill be described below.

CXCL12 and CXCR4 stimulate the phosphatidyl-inositol-3-kinase pathwaythat subsequently activates the protein kinase, Akt. Activated Aktphosphorylates a variety of intracellular targets, functioning toinhibit apoptosis and prolonging survival in different types of cancercells. Beyond cell survival, Akt has also been implicated in effects ofCXCR4 on migration of cells toward CXCL12 and their proliferation.

The mitogen-activated protein (MAP) kinase pathway is another signaltransduction pathway regulated by CXCR4. Following stimulation withCXCL12, CXCR4 activates the kinase MEK, which in turn activates ERK1/2MAP kinases. Activated ERK1/2 kinases phosphorylate transcriptionfactors such as Elk-1; this process increases expression of genes thatpromote survival and proliferation of cancer cells.

CXCR4 also appears to regulate angiogenesis, the process that isimportant for both normal physiology and growth of tumors. Mice lackingCXCR4 or CXCL12 have defective formation of blood vessels in thegastrointestinal tract. Pro-angiogenic effect of CXCR4 signaling may bemediated through up-regulation of vascular-endothelial growth factor(VEGF). Thus, another potential function of CXCR4 signaling in tumordevelopment is promotion of blood vessel production.

The CXCR4/CXCL12 Axis in Hematopoietic Stem Cell Mobilization

All mature blood cells are derived from hematopoietic stem cells (HSC)through intermediates that are termed hematopoietic progenitor cells(HPCs). Hematopoietic cells at various stages of differentiation arelocalized within the bone marrow (BM), their main site of production.Their mobilization between BM and blood is a physiological process, butunder steady-state conditions HPCs and HSCs circulate in the blood atfrequencies too low to allow for efficient collection of numberssufficient to transplantation. Recently, the use of peripheral blood assource of HSCs for transplantations has replaced bone marrow as thepreferred source of hematopoietic rescue. Stem cell frequencies in bloodare considerably increased both in responses to various growth factorsand during the recovery phase following myelosuppressive chemotherapy.Increased number of hematopoietic cells in the blood and amelioration oftheir mobilization ability will improve the efficiency oftransplantation and will shorten the time of cytopenia and engraftment.

Granulocyte Colony-stimulating Factor (G-CSF)-mobilized peripheral-bloodmononuclear cells are routinely used as a source of hematopoietic stemcells for transplantation. However, this mobilization results in broadinter-individual variations in circulating progenitor cell numbers.Thus, optimal methods to mobilize and collect peripheral-bloodprogenitor cells for hematopoietic rescue still need to be found.

Over recent years it has become apparent that the interaction betweenCXCL12 and its receptor, CXCR4, plays pivotal role in mobilization andengraftment of hematopoietic cells (Kollet et al., 2002; Lapidot et al.,2002; Levesque et al., 2003; Peled et al., 1999; Lapidot et al., 2005;Dar et al., 2005). The CXCR4 receptor is widely expressed on many celltypes including HSCs and HPCs and the interaction with its ligand seemsto be involved in their chemotaxis, homing and survival. TheCXCL12/CXCR4 axis was found to be involved in the retention ofhematopoietic cells within the bone marrow microenvironment (Kim et al.,1998) and consequently, it was suggested that antagonizing theinteractions of marrow-produced CXCL12 with CXCR4 expressed on HSCsmight be an effective HSC mobilizing strategy.

CXCR4 Modulators and T-140 Analogs

Various uses of chemokine receptor modulators, including CXCR4 agonistsand antagonists, have been described in the art (Princen et al., 2005;Tamamura et al., 2005). For example, the bicyclam drug termed AMD3100,originally discovered as an anti-HIV compound, specifically interactswith CXCR4 in an antagonistic manner. Blocking CXCR4 receptor withAMD3100 results in the mobilization of hematopoietic progenitor cells;when combining AMD3100 with G-CSF, additive effects were detected(Flomenberg et al., 2005; Broxmeyer et al., 2005). AMD3100 is currentlyundergoing clinical trials to evaluate its ability to increase stemcells available for transplant (Lack et al., 2005). U.S. Pat. No.6,365,583 discloses a method to treat a subject who would be benefitedby elevation of white blood cell count which method comprisesadministering to said subject a cyclic polyamine such as AMD3100. Martinet al. (2003) show that the mobilization of neutrophils from the bonemarrow by the CXCR2-chemokine, KC, was enhanced by AMD3100, examined 60minutes after administration to normal BALB/c mice.

U.S. Patent Application Publication No. 2004/0209921 disclosesheterocyclic compounds that bind to chemokine receptors, including CXCR4and CCR5, which may possess protective effects against infection oftarget cells by a human immunodeficiency virus (HIV). Other potentialuses for these compounds suggested by '921 are enhancing the populationof progenitor and/or stem cells, stimulating the production of whiteblood cells, and/or effecting regeneration of cardiac tissue.

U.S. Pat. No. 6,946,445 discloses CXCR4 antagonists comprising thesequence KGVSLSYR. The antagonists disclosed by the '445 patent aresuggested to be potentially useful for reducing interferon gammaproduction by T-cells, treatment of an autoimmune disease, treatment ofmultiple sclerosis, treatment of other neurological diseases, treatmentof cancer, and regulation of angiogenesis. U.S. Pat. No. 6,875,738discloses methods for treating a solid tumor in a mammal and forinhibiting angiogenesis in a mammal using these antagonists.

U.S. Patent Application Publication No. 2005/0002939 discloses a methodof treating ovarian cancer in a mammal, the method comprisingadministering to the mammal a therapeutically effective dose of a CXCR4inhibitor. The '939 application suggests that an anti-CXCR4 antibody mayimpact the survival or growth of a CXCR4-expressing tumor derived from abladder tumor cell line in a mouse model.

T-140 is a 14-residue synthetic peptide developed as a specific CXCR4antagonist that suppresses HIV-1 (X4-HIV-1) entry to T cells throughspecific binding to CXCR4 (Tamamura et al., 1998). Subsequently, peptideanalogs of T-140 were developed as specific CXCR4 antagonist peptideswith inhibitory activity at nanomolar levels (see Tamamura et al., 2003,WO 2002/020561 and WO 2004/020462).

WO 2002/020561 discloses novel peptide analogs and derivatives of T-140.The '561 publication demonstrates that the claimed peptides are potentCXCR4 inhibitors, manifesting high anti-HIV virus activity and lowcytotoxicity.

WO 2004/020462 discloses additional novel peptide analogs andderivatives of T-140, including 4F-benzoyl-TN14003 (SEQ ID NO:1). The'462 publication further discloses novel preventive and therapeuticcompositions and methods of using same utilizing T-140 analogs for thetreatment of cancer and chronic rheumatoid arthritis. The specificationof '462 demonstrates the ability of these peptides to inhibit cancercell migration, including breast cancer and leukemia cells, and toinhibit metastasis formation in vivo. Further demonstrated therein isinhibition of delayed-type hypersensitivity reaction in mice andcollagen-induced arthritis, an animal model of rheumatoid arthritis.

WO 2004/087068 is directed to a method for treating or preventing aCXCR4 mediated pathology comprising administering a CXCR4 peptideantagonist to a host in an amount sufficient to inhibit CXCR4 signaltransduction in a cell expressing a CXCR4 receptor or homologue thereof,wherein the CXCR4 peptide antagonist is not an antibody or fragmentthereof. The '068 publication discloses that exemplary CXCR4 peptideantagonists include T140 and derivatives of T140, and that the pathologyincludes cancer such as breast, brain, pancreatic, ovarian, prostate,kidney, and non-small lunch cancer. Other publications directed to theuse of CXCR4 antagonists in cancer therapy include, for example, WO00/09152, US 2002/0156034, and WO 2004/024178.

A recent publication by some of the inventors of the present invention(Avniel et al., 2006) discloses that blocking the CXCR4/CXCL12 axis by aT-140 analog resulted in a significant reduction in eosinophilaccumulation in the dermis and improved epithelialization, thussignificantly improving skin recovery after burns.

None of the prior art discloses or suggests that CXCR4 inhibitorpeptides belonging to the T-140 analog family may also affect CXCR4activity in an agonist manner. There exists a long felt need forcompositions and methods useful for modulating CXCR4-mediated processesinvolved in pathological conditions in vivo.

SUMMARY OF THE INVENTION

The present invention is directed to novel therapeutic applications ofT-140 analog peptides. The present invention discloses, for the firsttime, that T-140 analogs, hitherto known as CXCR4 inhibitors,unexpectedly also possess CXCR4 super-agonistic properties. The presentinvention thus provides compositions and methods utilizing T-140 analogsin applications in which activation of CXCR4 in an agonistic manner isbeneficial, such as for inducing tumor cell death in hematopoietic andglial malignancies.

The invention is also based, in part, on the unexpected discovery thatthe known T-140 analog 4F-benzoyl-TN14003(4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂,SEQ ID NO:1), but not the bicyclam CXCR4 inhibitor AMD3100, canselectively, specifically and rapidly stimulate multiple myeloma, gliomaand leukemia cell death, both in vitro and in vivo, thus demonstratingincreased anti-tumor effects particularly on tumors of hematopoietic andglial origin. Unexpectedly, 4F-benzoyl-TN14003 was also found tosynergize with rapamycin in inducing multiple myeloma cell death.

According to certain embodiments of the invention, the4F-benzoyl-TN14003 and analogs and derivatives thereof are now disclosedto be particularly useful in the treatment of hematopoietic and glialtumors. Specifically, there is provided a method for treating a subjecthaving a tumor selected from the group consisting of multiple myeloma,microglioma and glioma, comprising administering to the subject atherapeutically effective amount of a peptide having an amino acidsequence as set forth in SEQ ID NO:1 or an analog or derivative thereof.

The 4F-benzoyl-TN14003 analogs and derivatives used in the novelcompositions and methods of the invention are the structurally andfunctionally related peptides disclosed in patent applications WO2002/020561 and WO 2004/020462, also known as “T-140 analogs”, asdetailed hereinbelow.

In various particular embodiments, the analog or derivative has an aminoacid sequence as set forth in the following formula (I) or a saltthereof:

(I) 1  2  3  4   5  6  7 8  9 10 11 12 13  14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁wherein:

-   -   A₁ is an arginine, lysine, ornithine, citrulline, alanine or        glutamic acid residue or a N-α-substituted derivative of these        amino acids, or A₁ is absent;    -   A₂ represents an arginine or glutamic acid residue if A₁ is        present, or A₂ represents an arginine or glutamic acid residue        or a N-α-substituted derivative of these amino acids if A₁ is        absent;    -   A₃ represents an aromatic amino acid residue;    -   A₄, A₅ and A₉ each independently represents an arginine, lysine,        ornithine, citrulline, alanine or glutamic acid residue;    -   A₆ represents a proline, glycine, ornithine, lysine, alanine,        citrulline, arginine or glutamic acid residue;    -   A₇ represents a proline, glycine, ornithine, lysine, alanine,        citrulline or arginine residue;    -   Ag represents a tyrosine, phenylalanine, alanine,        naphthylalanine, citrulline or glutamic acid residue;    -   A₁₀ represents a citrulline, glutamic acid, arginine or lysine        residue;    -   A₁₁ represents an arginine, glutamic acid, lysine or citrulline        residue wherein the C-terminal carboxyl may be derivatized;        and the cysteine residue of the 4-position or the 13-position        can form a disulfide bond, and the amino acids can be of either        L or D form.

Exemplary peptides according to formula (I) are peptides having an aminoacid sequence as set forth in any one of SEQ ID NOS:1-72, as presentedin Table 1 hereinbelow.

In another preferable embodiment, the analog or derivative has an aminoacid sequence as set forth in SEQ ID NO:65(H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;TC14003).

In certain other particular embodiments, said analog or derivative isselected from the group consisting of:

(SEQ ID NO: 1) 4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂, (SEQ ID NO: 2)Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg- Cit-Cys-Arg-OH,(SEQ ID NO: 3) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH, (SEQ ID NO: 4)Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg- Cit-Cys-Arg-OH,(SEQ ID NO: 10) Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂, (SEQ ID NO: 46)TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro- Tyr-Arg-Cit-Cys-Arg-NH₂;,(SEQ ID NO: 47) ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂, (SEQ ID NO: 51)Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg- Cit-Cys-Arg-NH₂,(SEQ ID NO: 52) Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂, (SEQ ID NO: 53)4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHMe, (SEQ ID NO: 54)4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHEt, (SEQ ID NO: 55)4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NHiPr, (SEQ ID NO: 56)4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-tyramine, (SEQ ID NO: 65)H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg- Cit-Cys-Arg-OH,(SEQ ID NO: 66) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ (SEQ ID NO: 68)H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg- Cit-Cys-Arg-NH₂,(SEQ ID NO: 70) H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH, and (SEQ ID NO: 71)H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg- Cit-Cys-Arg-OH.

In another aspect, there is provided a method for inducing hematopoietictumor cell death in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a peptide having anamino acid sequence as set forth in SEQ ID NO:1 or an analog orderivative thereof. In another particular embodiment, the tumor isselected from leukemia, lymphoma, microglioma and multiple myeloma. In aparticular embodiment, the tumor is multiple myeloma.

In another aspect, there is provided a method for inducing glial tumorcell death in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a peptide having an aminoacid sequence as set forth in SEQ ID NO:1 or an analog or derivativethereof.

Without wishing to be bound by any theory or mechanism of action, theT-140 analogs of the invention are useful for inducing tumor cellapoptosis.

Other embodiments of the present invention are directed to the use of4F-benzoyl-TN14003 or an analog or derivative thereof for sensitizingtumors to chemotherapeutic or other anti-cancer drugs.

Thus, in another aspect, there is provided a method for increasing thesensitivity of tumor cells to an anti-cancer agent in a subject in needthereof comprising administering to the subject a sensitizing-effectiveamount of a peptide having an amino acid sequence as set forth in SEQ IDNO:1 or an analog or derivative thereof in concurrent or sequentialcombination with the anti-cancer agent.

In another embodiment, the anti-cancer agent is a chemotherapeutic drug.For example, the chemotherapeutic drug may be selected from alkylators(e.g. cyclophosphamide and isophosphamide and platinum-containingcompounds), anthracyclines, antibiotics (e.g. adreamaycin), aromataseinhibitors, bisphosphonates, cyclo-oxygenase inhibitors, estrogenreceptor modulators, folate antagonists, inorganic aresenates,microtubule inhibitors (e.g. taxanes), nitrosoureas, nucleoside analogs,osteoclast inhibitors, retinoids, proteasome inhibitors (e.g. Velcade),topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, antimetabolites(e.g. Methotrexate) and tyrosine kinase inhibitors.

In another particular embodiment, the drug is an apoptosis-inducingdrug. In a preferable embodiment, the drug is rapamycin or a derivativethereof.

In another particular embodiment, the drug is an immunosuppressant usedto treat hematopoietic tumors such as corticosteroids (e.g.dexamethasone) and immunosuppressive antibodies (e.g. Mabthera).

In another particular embodiment, the tumor is a hematopoietic tumor(e.g. leukemia, lymphoma, multiple myeloma and microglioma).

In another particular embodiment, the tumor is a glial tumor (e.g.ependymomas, astrocytomas, oligodendrogliomas and mixed gliomas, such asoligoastrocytomas).

In another aspect, the invention provides a pharmaceutical compositioncomprising as active ingredients a peptide having an amino acid sequenceas set forth in SEQ ID NO:1 or an analog or derivative thereof andrapamycin or a derivative thereof.

The combinations of the invention may also be in form of a kit or apharmaceutical pack containing one or more courses of treatment for aneoplasm in a subject in need thereof. Thus, there is provided inanother aspect a kit containing i) a peptide having an amino acidsequence as set forth in SEQ ID NO:1 or an analog or derivative thereofand ii) a chemotherapeutic agent comprising rapamycin or a derivativethereof, and optionally iii) instructions for administering said peptideand said chemotherapeutic agent to a subject in need thereof, e.g. to asubject afflicted with a hematopoietic or glial tumor.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 demonstrates that 4F-benzoyl-TN14003 enhances MIP3α secretion bythe prostate cancer cell line PC3 in a CXCR4-dependent manner. FIG. 1Aillustrates FACS analysis of PC3 cells (left panel) and PC3-CXCR4.5cells (right panel) stained for the control and CXCR4 antibodies. FIG.1B illustrates MIP3α secretion, assessed by ELISA, of PC3 cells (leftpanel) and PC3-CXCR4.5 cells (right panel) stimulated with differentconcentrations of CXCL12 for 48 hours. FIG. 1C illustrates MIP3αsecretion, assessed by ELISA, of PC3 cells (left panel) and PC3-CXCR4.5cells (right panel) treated with anti-CXCL12 antibodies, Pertussistoxin, AMD3100 or 4F-benzoyl-TN14003, alone or in combination withCXCL12.

FIG. 2 depicts the effect of 4F-benzoyl-TN14003 on proliferation ofnormal hematopoietic cells and primary keratinocytes. FIG. 2A,proliferation of human CD4+ T cells in response to increasing doses ofCXCL12, or in response to the treatment with the CXCR4 antagonistAMD-3100 and 4F-benzoyl-TN14003. FIG. 2B, proliferation of human CD34+hematopoietic stem cells. FIG. 2C, proliferation of human primarykeratinocytes.

FIG. 3 illustrates the effect of AMD-3100 and 4F-benzoyl-TN14003 on theproliferation of prostate carcinoma PC3 and PC3-CXCR4.5 cells. FIG. 3A,proliferation of PC3 cells in response to increasing doses of CXCL12, orin response to the treatment with a single dose of AMD-3100 and4F-benzoyl-TN14003. FIG. 3B, proliferation of PC3-CXCR4.5 cells inresponse to increasing doses of CXCL12, or in response to the treatmentwith a single dose of AMD-3100 and 4F-benzoyl-TN14003. FIG. 3C,proliferation of PC3 cells in response to increasing doses of4F-benzoyl-TN14003. FIG. 3D, proliferation of PC3-CXCR4.5 cells inresponse to increasing doses of 4F-benzoyl-TN14003. The resultsrepresent average of triplicates±STDEV.

FIG. 4 demonstrates that cancer cell lines of epithelial origin, SKBR3(breast carcinoma), U87 (glioblastoma) and 22RV1 (prostate carcinoma) donot demonstrate significant response in proliferation followingstimulation with CXCL12, AMD-3100 or 4F-benzoyl-TN14003. Proliferationof cancer cell lines, SKBR3 (FIG. 4A), 22RV1 (FIG. 4B) and U87 (FIG.4C), in response to increasing doses of CXCL12, or in response to thetreatment with a single dose of CXCR4 antagonists, AMD-3100 and4F-benzoyl-TN14003. The results represent average of triplicates±STDEV.

FIG. 5 shows the effect of 4F-benzoyl-TN14003 on proliferation of K562CML cells. FIG. 5A, proliferation of human CML K562 cells that expresslow (gray columns) and high (black columns) levels of CXCR4 before(control) and after treatment with increasing doses of the CXCR4antagonist AMD-3100 (25 μM) and 4F-benzoyl-TN14003 (4-20 μM). Theresults represent average of triplicates±STDEV. FIG. 5B, the percentageof apoptotic annexin positive cells determined by FACS of K562 cells orK562 cells overexpressing CXCR4 (“K562-CXCR4”).

FIG. 6 illustrates the effect of 4F-benzoyl-TN14003 on proliferation ofHL-60 and NB4 AML cells. FIGS. 6A and 6C depict the results of differentexperiments measuring proliferation of human HL-60 cells before(control) and after treatment with increasing doses of the CXCR4antagonist AMD-3100 (25 μM) and 4F-benzoyl-TN14003 (4-20 μM). Theresults represent average of triplicates±STDEV. FIG. 6B, the percentageof apoptotic annexin positive cells determined by FACS. The effect of4F-benzoyl-TN14003 on the survival of HL-60 and NB-4 cells can beinhibited by pretreatment of cells with the CXCR4 antagonist AMD-3100.FIG. 6D, proliferation of human NB4 cells before and after treatmentwith increasing doses of the CXCR4 antagonist AMD-3100 (25 μM) and4F-benzoyl-TN14003 (4-20 μM).

FIG. 7 depicts the Effect of 4F-benzoyl-TN14003 on the proliferation ofRPMI8226, ARH77, U266, and NCI multiple myeloma (MM) cells.Proliferation of human RPMI8226 (FIG. 7A), ARH77 (FIG. 7C), U266 (FIG.7E), NCI (FIG. 7G) MM cells before (control) and after treatment withincreasing doses of the CXCR4 antagonists AMD-3100 (20 μM) and4F-benzoyl-TN14003 4-20 μM). The results represent average oftriplicates±STDEV. The percentage of apoptotic annexin positive cellswas determined for each cell line by FACS (FIGS. 7B, 7D, 7F and 7H,respectively).

FIG. 8 demonstrates that the effect of 4F-benzoyl-TN14003 on theproliferation of RPMI8226MM cells and migration of T cell leukemiaJurkat cell is abolished upon treatment with Proteinease K. FIG. 8A,inhibition of the effect of 4F-benzoyl-TN14003 on proliferation of humanRPMI8226 following treatment of the peptide with Proteinease K. FIG. 8B,inhibition of the effect of 4F-benzoyl-TN14003 on apoptosis of humanRPMI8226 following treatment of the peptide with Proteinease K. FIG. 8C,inhibition of the inhibitory effect of 4F-benzoyl-TN14003 on migrationof Jurkat cells in response to CXCL12 following treatment of the peptidewith Proteinease K.

FIG. 9 illustrates the effect of 4F-benzoyl-TN14003 on the proliferationand survival of RPMI8226MM cells over time. FIG. 9A, the effect of4F-benzoyl-TN14003 on the number of cells. FIG. 9B, the number ofapoptotic cells after 1 h of incubation with the peptide. FIG. 9C, thenumber of apoptotic cells after 4 h. FIG. 9D, the number of apoptoticcells after 24 h. FIG. 9E, the number of apoptotic cells after 72 h.

FIG. 10 depicts the effect of 4F-benzoyl-TN14003 on apoptosis ofRPMI8226MM cells. FIG. 10A indicates the different stages of apoptosisand necrosis. FIG. 10B shows untreated cells, and FIG. 10C showsRPMI8226 cells undergoing cell death in response to 4F-benzoyl-TN14003(8 μM).

FIG. 11 illustrates the effect of 4F-benzoyl-TN14003 on theproliferation and survival of BM derived primary MM cells. The effect of4F-benzoyl-TN14003 on the survival of BM derived cells from MM patientswith different percentage of CD138+ MM cells. FIGS. 11A and 11C, 100% MMcells. FIG. 11B, 80% MM cells. FIG. 11D, 5% MM cells.

FIG. 12 demonstrates the effect of 4F-benzoyl-TN14003 on the survival ofBM derived MM CD138 and CD34 positive cells. FIG. 12A, the effect of4F-benzoyl-TN14003 on the survival of BM derived CD138⁺ cells (R3). FIG.12B, the effect of 4F-benzoyl-TN14003 on the survival of BM derivedCD34⁺ cells (R2). Left panels, untreated cells; right panels,4F-benzoyl-TN14003-treated cells.

FIG. 13 presents the effect of Rapamycin on the proliferation andsurvival of RPMI8226MM cells. Diamonds represent untreated cells(“ctrl”); squares represent cells treated with 10 μM rapamycin (“Rapa 10μM”); triangles represent cells treated with 20 μM rapamycin; circlesrepresent cells treated with 50 μM rapamycin; and crosses representcells treated with 1000 μM rapamycin.

FIG. 14 depicts the effect of rapamycin in combination with4F-benzoyl-TN14003 on the proliferation and survival of RPMI8226MMcells.

FIG. 15 presents the effect of 4F-benzoyl-TN14003 on the proliferationand survival of U87 glioma cells. FIG. 15A, the effect of4F-benzoyl-TN14003 on cell survival. FIG. 15B, the effect of4F-benzoyl-TN14003 on cell death.

FIG. 16 shows the effect of i.p. injection of 4F-benzoyl-TN14003 (8mg/Kg) on NB4 derived tumor growth.

FIG. 17 presents the effect of s.c. injection of 4F-benzoyl-TN14003 (8mg/Kg) on NB4, derived tumor growth. FIG. 17A, effect on tumor size.FIG. 17B, effect on tumor weight.

FIG. 18 demonstrates detection of K562L cells in vivo using the CCCDcamera, 24 hr after IP injection. FIG. 18A, 2.5×10⁵ injected cells. FIG.18B, 5×10⁵ injected cells. FIG. 18C, 10⁶ injected cells. FIG. 18D, 2×10⁶injected cells.

FIG. 19 demonstrates that STI571 inhibits tumor development of K562L invivo over time. STI571 (12 mg/Kg, or 40 mg/Kg per mouse), which inhibitsselectively the tyrosine-kinase activity of c-abl, was injected togetherwith K562L cells (2×10⁶/mice). Mice were further treated with STI571 ondays 3, and 5 following injection of the cells and the amount of lightemission was evaluated using the CCCD camera on Days 1, 4 and 7 aftercell injection.

FIG. 20 demonstrates that STI571 and 4F-benzoyl-TN14003 inhibit tumordevelopment of K562L cells in vivo. STI571 (40 mg/Kg) and4F-benzoyl-TN14003 (4 mg/Kg), were injected IP with K562L cells. Thedevelopment of tumors was evaluated 24 hr later using the CCCD camera.Mice were further treated with STI571 and 4F-benzoyl-TN14003 on days 3and 5 and the amount of light they emitted was evaluated using the CCCDcamera on days 5 and 8 after injection of the cells.

FIG. 21 shows that 4F-benzoyl-TN14003 inhibits tumor development ofK562L cells in vivo. 4F-benzoyl-TN14003 (2 mg/Kg) and AMD-3100 (4 mg/Kg)were injected i.p on days 2, 5, 7, 11 following injection of K562Lcells. The development of tumors was evaluated on day 17 using the CCCDcamera.

FIG. 22 demonstrates that 4F-benzoyl-TN14003 inhibits MM cell growth invivo. FIG. 22A, reduction of RPMI8226 tumor size upon s.c.administration of 4F-benzoyl-TN14003. FIG. 22B, reduction of tumor sizein established RPMI8226 tumors upon s.c. administration of4F-benzoyl-TN14003.

FIG. 23 demonstrates that 4F-benzoyl-TN14003 stimulates apoptotic MMcell death in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel compositions and methodswherein T-140 analog peptides, hitherto known as CXCR4 antagonists, areused to stimulate CXCR4-mediated processes in an agonistic manner.

The present invention discloses for the first time that4F-benzoyl-TN14003 (SEQ ID NO:1), a known CXCR4 inhibitor belonging tothe T-140 peptide family, mediates unique beneficial effects, which arenot mediated by other CXCR4 inhibitors such as AMD3100. Unexpectedly,4F-benzoyl-TN14003 was found to induce apoptosis in hematopoietic tumorssuch as glioma and multiple myeloma, and to enhance tumor cell apoptosisinduced by rapamycin.

T-140 Analogs

The peptides described in this specification have an N-terminus(amino-terminal) at the left extremity and a C-terminus(carboxyl-terminal) at the right extremity in accordance with thecustomary practice of peptide notations.

In this specification and drawings, the representations of amino acids,etc. by brevity codes are made by the use of the codes prescribed byIUPAC-IUB Commission on Biochemical Nomenclature or by the codescustomarily used in the relevant art. Examples of such codes are shownbelow. If an optical isomer exists with respect to an amino acid, itpreferably represents the L form unless otherwise expressly specified.

Gly or G: glycine; Ala or A: alanine; Val or V: valine; Leu or L:leucine; Ile or I: isoleucine; Ser or S: serine; Thr or T: threonine;Cys or C: cysteine; Met or M: methionine; Glu or E: glutamic acid; Aspor D: aspartic acid; Lys or K: lysine; Arg or R: arginine; H is or H:histidine; Phe or F: phenylalanine; Tyr or Y: tyrosine; Trp or W:tryptophan; Pro or P: proline; Asn or N: asparagine; Gln or Q:glutamine; pGlu: pyroglutamic acid; Nal: 3-(2-naphthyl) alanine; Cit:citrulline; DLys: D-lysine; DCit: D-citrulline; DGlu: D-glutamic acid;Me: methyl group; Et: ethyl group; Bu: butyl group; Ph: phenyl group.

The substituents, protective group and reagents often used in thisspecification are indicated by the following codes.

BHA: benzhydrylamine

pMBHA: p-methylbenzhydrylamine

Tos: p-toluenesulphonyl

CHO: formyl

HONB: N-hydroxy-5-norbornene-2,3-dicarboximide

OcHex: cyclohexyl ester

Bzl: benzyl

Cl₂-Bzl: dichloro-benzyl

Bom: benzyloxymethyl

Z: benzyloxycarbonyl

Br—Z: 2-bromobenzyloxycarbonyl

Boc: t-butyloxycarbonyl

DCM: dichloromethane

HOBt: 1-hydroxybenzotriazole

DCC: N,N′-dicyclohexylcarbodiimide

TFA: trifluoroacetic acid

DIEA: diisopropylethylamine

Fmoc: N-9-fluorenylmethoxycarbony

DNP: dinitrophenyl

Bum: tertiarybutoxymethyl

Trt: trityl

Ac: acetyl

Guanyl: guanyl

Succinyl: succinyl

glutaryl: glutaryl

TMguanyl: tetramethylguanyl

2F-benzoyl: 2-fluorobenzoyl

4F-benzoyl: 4-fluorobenzoyl

APA: 5-aminopentanoyl

ACA: 6-aminohexanoyl

desamino-Arg: 2-desamino-arginyl

deamino TMG-APA: the following formula (IV):

R—CH2: the following formula (V):

In N-terminal amino acids, [H—] indicates that the terminal amino groupis not derivatized, and in C-terminal amino acids, [—OH] indicates thatthe terminal carboxyl group is not derivatized.

The 4F-benzoyl-TN14003 analogs and derivatives of the invention belongto a family of structurally closely related peptides, also known asT-140 analogs.

T-140 is a known peptide having the amino acid sequenceH-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH (SEQ IDNO:69, Tamamura et al., 2003). The preferable peptides of the inventioninclude analogs and derivatives disclosed in patent applications WO2002/020561 and WO 2004/020462.

In one aspect, the present invention relates to the use ofpharmaceutical compositions comprising as an active ingredient a peptideindicated by the following formula (I) or a salt thereof:

(I) 1  2 3   4   5  6  7 8  9 10 11 12 13  14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁wherein:

A₁ in the above-mentioned formula (I) represents an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue (either L or Dform) which may be derivatized at the N-terminus, or A₁ is a hydrogenatom, or it is preferable that A₁ is an arginine, citrulline, alanine orD-glutamic acid residue, or A₁ is a hydrogen atom.

Examples of “N-terminal derivatized peptides” or “N-α-substitutedderivatives” include, but are not limited to, those protected by formylgroup; acyl group, e.g., acetyl group, propionyl group, butyryl group,pentanoyl group, C2-6alkanoyl group e.g. hexanoyl group, benzoyl group,arylcarbonyl group e.g. substituted benzoyl group (e.g.:2-fluorobenzoyl, 3-fluorobenzoyl group, 4-fluorobenzoyl group,2-bromobenzoyl group, 3-bromobenzoyl group, 4-bromobenzoyl group,2-nitrobenzoyl group, 3-nitrobezoyl group, 4-nirtobenzoyl group),succinyl group, glutaryl group; nicotinyl group; isonicotinyl group;alkylsulfonyl group (e.g.: methanesulfonyl group, ethanesulfonyl group,propanesulfonyl group, camphorsulfonyl group); arylsulfonyl group (e.g.:p-toluenesulfonyl group, 4-fluorobenzenesulfonyl group,mesitylenesulfonyl group, 4-aminobenzenesulfonyl group, dansyl group,4-bromobenzenesulfonyl group) etc. Or, the amino acid group ofN-terminal may be absent.

Optionally and preferably, the peptide is derivatized at the N terminuswith a substituted benzoyl group. In a particular embodiment, thesubstituted benzoyl group is a 4-fluorobenzoyl group. In anotherparticular embodiment, the substituted benzoyl group is a2-fluorobenzoyl group.

A₂ in the above-mentioned formula (I) represents an arginine or glutamicacid residue (either L or D form) if A1 is an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue (either L or Dform) which may be derivatized at the N-terminal, or A₂ represents anarginine or glutamic acid residue (either L or D form) which may bederivatized at the N-terminus if A₁ is absent, or it is preferable thatA₂ is an arginine or glutamic acid residue if A₁ is an arginine,citrulline, alanine or glutamic acid residue which may be derivatized atthe N-terminal, or A₂ is an arginine or glutamic acid residue which maybe derivatized at N-terminal if A₁ is absent. Examples of “peptidesderivatized at the N-terminal” include, but are not limited to, the sameones as those mentioned in A1.

A₃ in the above-mentioned formula (I) represents an aromatic amino acidresidue (e.g., phenylalanine, tryptophan, 3-(2-naphthyl)alanine,tyrosine, 4-fluorophenylalanine, 3-(1-naphthyl)alanine (either L or Dform), or preferably, A₃ represents phenylalanine, tryptophan or3-(2-naphthyl)alanine.

A₄ in the above-mentioned formula (I) represents an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue (either L or Dform), or it is preferable that A₄ is an arginine, citrulline, alanineor L- or D-glutamic acid residue.

A₅ in the above-mentioned formula (I) represents an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue (either L or Dform), or it is preferable that A₅ is an arginine, citrulline, alanine,lysine or glutamic acid residue.

A₆ in the above-mentioned formula (I) represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue (either L or D form), or it is preferable that A₆ is a D-lysine,D-alanine, D-citrulline or D-glutamic acid residue.

A₇ in the above-mentioned formula (I) represents a proline, glycine,ornithine, lysine, alanine, citrulline or arginine residue (either L orD form), or it is preferable that A₇ is a proline or alanine residue.

A₈ in the above-mentioned formula (I) represents a tyrosine,phenylalanine, alanine, naphthylalanine, citrulline or glutamic acidresidue (either L or D form), or it is preferable that A₈ is a tyrosine,alanine or D-glutamic acid residue.

A₉ in the above-mentioned formula (I) represents an arginine, lysine,ornithine, citrulline, alanine or glutamic, acid residue (either L or Dform), or it is preferable that A₉ is an arginine, citrulline orglutamic acid residue.

A₁₀ in the above-mentioned formula (I) represents a citrulline, glutamicacid, arginine or lysine residue (either L or D form), or it ispreferable that A₁₀ is a citrulline or D-glutamic acid residue.

A₁₁ in the above-mentioned formula (I) represents an arginine, glutamicacid, lysine or citrulline residue (either L or D form) which may bederivatized at C-terminal, or it is preferable that A₁₁ is an arginineor glutamic acid residue which may be derivatized at C-terminal.

“C-terminal derivatization” or “C-terminal carboxyl derivatization”includes, without limitation, amidation (—CONH₂, —CONHR, —CONRR′) andesterification (—COOR). Herein, R and R′ in amides and esters include,for example, C₁₋₆ alkyl group e.g. methyl, ethyl, n-propyl, isopropyl,or n-butyl, C₃₋₈ cycloalkyl group e.g. cyclopentyl, cyclohexyl, C₆₋₁₂aryl group e.g. phenyl and a-naphthyl, phenyl-C₁₋₂ alkyl group e.g.benzyl, phenethyl or C₇₋₁₄ aralkyl group e.g. C₁₋₂ alkyl group e.g.a-naphthyl methyl group, and additionally, pivaloyloxymethyl group whichis generally used as an oral bioavailable ester.

If a peptide of the present invention has carboxy groups (orcarboxylates) at side-chain terminals other than C-terminal, the peptidehaving amidated or esterificated carboxy groups at side-chain terminalsis included in the peptides of the present invention. As the amides andesters in this case, for example, the amides and esters exemplified inA₁₁ are similarly used. Also, the peptides of the present inventioninclude peptides in which substituents (e.g. —OH, —SH, amino group,imidazole group, indole group, guanidino group, etc.) on theintramolecular amino acid side chains are protected by suitableprotective group (e.g. C1-6 acyl group, C2-6 alkanoyl such as formylgroup, acetyl group, etc.), or complex peptides such as glycopeptidescombined with sugar chain in the above-mentioned peptides.

Salts of the peptides of the present invention include physiologicallyacceptable salts of acids or bases and particularly, physiologicallyacceptable acid addition salts are preferable. Such salts areexemplified by salts of inorganic acids (e.g. hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid), or salts of organicacids (e.g. acetic acid, formic acid, propionic acid, fumaric acid,maleic acid, succinic acid, tartaric acid, citric acid, malic acid,oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

In one embodiment, the composition comprises a peptide as set forth informula (I) as defined hereinabove, wherein A₁ is a glutamic acidresidue or is absent.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₄ is a glutamic acidresidue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₆ is a glutamic acidresidue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₈ is a glutamic acidresidue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₉ is a glutamic acidresidue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₅ is an arginine orglutamic acid residue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₁₀ is a glutamic acid,arginine or lysine residue.

In another embodiment, the composition comprises a peptide as set forthin formula (I) as defined hereinabove, wherein A₁₁ is a glutamic acid,lysine or citrulline residue.

In another embodiment, the peptide has an amino acid sequence as setforth in any one of SEQ ID NOS:1-72 presented in Table 1 herein:

TABLE 1 T-140 and currently preferred T-140 analogs SEQ ID Analog NO:Amino acid sequence 4F-benzoyl- 14F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys- TN14003Arg-NH₂ AcTC14003 2Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC140053 Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OHAcTC14011 4Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC140135 Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OHAcTC14015 6Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC140177 Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OHAcTC14019 8Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH AcTC140219 Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OHAcTC14012 10Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14014 11Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂AcTC14016 12Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14018 13Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14020 14Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂AcTC14022 15Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂ TE1400116 H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14002 17 H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14003 18 H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14004 19 H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14005 20 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14006 21 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OHTE14007 22 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OHTE14011 23H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE1401224 H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14013 25 H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE14014 26H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE1401527 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH₂TE14016 28 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH₂ AcTE14014 29Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTE14015 30Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg- NH₂AcTE14016 31Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg- NH₂ TF1:32 Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTE14011 TF2: guanyl- 33guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg- TE14011NH₂ TF3: 34TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys- TMguanyl-Arg-NH₂ TE14011 TF4: 35TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg- TMguanyl-NH₂ TE14011 (2- 14) TF5: 4F- 364F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-benzoyl- Arg-NH₂ TE14011 TF6: 2F- 372F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-benzoyl- Arg-NH₂ TE14011 TF7: APA- 38APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE14011 (2-14) TF8: 39desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-desamino-R- NH₂ TE14011 (2- 14) TF9: guanyl- 40Guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2- 14) TF10: 41succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂succinyl- TE14011 (2- 14) TF11: 42glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂glutaryl- TE14011 (2- 14) TF12: 43deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit- deaminoTMCys-Arg-NH₂ G-APA- TE14011 (2- 14) TF15: H- 44R-CH2-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂Arg-CH2NH- RTE14011 (2-14) TF17: 45H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE14011 (2-14) TF18: 46TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys- TMguanyl-Arg-NH₂ TC14012 TF19: ACA- 47ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg- TC14012NH₂ TF20: ACA- 48ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg- T140 OHTZ14011 49H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ AcTZ1401150 Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTN14003 51Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTN14005 52Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂4F-benzoyl- 534F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-TN14011-Me Arg-NHMe 4F-benzoyl- 544F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-TN14011-Et Arg-NHEt 4F-benzoyl- 554F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-TN14011-1Pr Arg-NHiPr 4F-benzoyl- 564F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-TN14011- Arg-tyramine tyramine TA14001 57H-Ala-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14005 58H-Arg-Arg-Nal-Cys-Tyr-Ala-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14006 59H-Arg-Arg-Nal-Cys-Tyr-Arg-Ala-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14007 60H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DAla-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14008 61H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Ala-Tyr-Arg-Cit-Cys-Arg-OH TA14009 62H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Ala-Arg-Cit-Cys-Arg-OH TA14010 63H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Ala-Cit-Cys-Arg-OH TC14001 64H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC14003 65H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TN14003 66H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TC1400467 H-Arg-Arg-Nal-Cys-Tyr-Arg-Cit-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401268 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ T-14069 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401170 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1400571 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401872 H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂

In each one of SEQ ID NOS:1-72, two cysteine residues are preferablycoupled in a disulfide bond.

Currently preferred peptides according to the present invention arepeptides having an amino acid sequence as set forth in any one of SEQ IDNOS:1-72. More preferably, it has been previously reported that theT-140 derivatives having an amino acid sequence as set forth in any oneof SEQ ID NOS: 1-68 and 70-71 presented in Table 1 may have improvedstability in serum and reduced cytotoxicity relative to T-140 (SEQ IDNO:69). However, T-140 may be suitable for use in the methods of thepresent invention, e.g. when applied by local administration routes.

In another preferable embodiment, the peptide used in the compositionsand methods of the invention consists essentially of an amino acidsequence as set forth in SEQ ID NO: 1. In another preferable embodiment,the peptide used in the compositions and methods of the invention is ofan amino acid sequence as set forth in SEQ ID NO:1. In anotherembodiment, the peptide is at least 60%, preferably at least 70% andmore preferably at least 80% homologous to SEQ ID NO:1. In anotherembodiment, the peptide is at least about 90% homologous to SEQ ID NO:1.In another embodiment, the peptide is at least homologous to SEQ IDNO:1. In another embodiment, the peptide is at least about 95%homologous to SEQ ID NO:1. Each possibility represents a separateembodiment of the present invention.

In various other particular embodiments, the peptide is selected fromSEQ ID NOS:1-72, wherein each possibility represents a separateembodiment of the present invention.

In another particular embodiment, said peptide has an amino acidsequence as set forth in any one of SEQ ID NOS:1-4, 10, 46, 47, 51-56,65, 66, 68, 70 and 71. In another particular embodiment, said peptidehas an amino acid sequence as set forth in any one of SEQ ID NOS:4, 10,46, 47, 68 and 70. In another particular embodiment, said peptide has anamino acid sequence as set forth in any one of SEQ ID NOS:1, 2, 51, 65and 66. In another particular embodiment, said peptide has an amino acidsequence as set forth in any one of SEQ ID NOS:53-56.

In a preferable particular embodiment, said peptide has an amino acidsequence as set forth in SEQ ID NO: 1. In another particular embodiment,said peptide has an amino acid sequence as set forth in SEQ ID NO:2. Inanother particular embodiment, said peptide has an amino acid sequenceas set forth in SEQ ID NO:51. In another particular embodiment, saidpeptide has an amino acid sequence as set forth in SEQ ID NO:66.

In another aspect, the invention relates to the use of a pharmaceuticalcomposition comprising a peptide indicated by the following formula (II)or a salt thereof:

(II) 1   2  3  4   5  6  7 8  9 10 11  12 13A₁-Arg-A₂-Cys-Tyr-A₃-A₄-X-A₅-A₆-Cit-Cys-A₇

wherein:

A₁ represents a hydrogen atom, or an arginine, lysine, ornithine,citrulline or alanine residue or a N-α-substituted derivative of theseamino acids;A₂ represents an aromatic amino acid residue;A₃, A₄ and A₆ each independently represent an arginine, lysine,ornithine, citrulline or alanine residue;A₅ represents a tyrosine, phenylalanine, alanine, naphthylalanine orcitrulline residue;A₇ represents a lysine or arginine residue in which a carboxyl group maybe amidated or esterified;X is selected from the group consisting of:

-   (i) a peptide residue represented by the following formula (III):

(III)  l′ 2′ 3′  4′ 5′ 6′ -A₈-A₉-A₁₀-Gly-A₁₁-A_(l2)-

-   -   wherein A₈ and A₁₂ each independently represents an alanine,        valine, leucine, isoleucine, serine, cysteine or methionine        residue;    -   A₉ represents an aromatic amino acid residue, A₁₀ is selected        from the same amino acid residues as in A₃, A₁₁ represents a        tyrosine, phenylalanine, tryptophan, alanine, valine, leucine,        isoleucine, serine, cysteine or methionine residue, provided        that when both of the 1′-position and the 6′-position are        cysteine residues, they may be bonded in a disulfide bond,

-   (ii) a peptide selected from the group consisting of a    D-ornithyl-proline, prolyl-D-ornithine, D-lysyl-proline,    prolyl-D-lysine, D-arginyl-proline, prolyl-D-arginine,    D-citrullyl-proline, D-citrullyl-alanine, D-alanyl-citrulline,    prolyl-D-citrulline, glycyl-ornithine, ornithyl-glycine,    glycyl-lysine, lysyl-glycine, glycyl-arginine, arginyl-glycine,    glycyl-citrulline, citrullyl-glycine, D-alanyl-proline, and    D-lysyl-alanine,    -   and a hydrogen atom of a side chain ω-amino group of D-arginine,        L-arginine, D-lysine, L-lysine, D-ornithine or L-ornithine which        are constitutional amino acids of said peptide residues may be        substituted by a ω-aminoacyl group,    -   and the peptide residues of (i) and (ii) represent a peptide        residue which binds amino acid residues at the 7-position and        the 9-position through a peptide bond;    -   and the cysteine residues at the 4-position and the 12-position        may be bonded in a disulfide bond;        provided that, in the above polypeptide or a salt thereof,        either of the amino acid residues of A₁, A₃, A₄, A₅, A₆ and A₇        is an alanine or citrulline residue; or

-   (iii) a peptide residue containing a D-citrulline, D-alanine,    citrulline, or alanine residue) or a salt thereof.

In the polypeptides of the formula (II) of the present invention, A₁ ispreferably an arginine, alanine or citrulline residue; A₂ is preferablya tryptophan or naphthylalanine residue; A₃ is preferably arginine,alanine or citrulline residue; A₄ is preferably a lysine, alanine orcitrulline residue; X is preferably a D-lysyl-proline, D-alanyl-proline,D-lysyl-alanine or D-citrullyl-proline residue; A₅ is preferably atyrosine or alanine residue; A₆ is preferably an arginine, alanine orcitrulline residue; A₇ is preferably an arginine residue.

Exemplary peptides of the formula (II) are peptides wherein A₁, A₆ andA₇ are arginine residues, A₂ is a naphthylalanine residue, A₃ is acitrulline residue, A₄ is a lysine residue, X is a D-lysyl-prolineresidue, and A₅ is a tyrosine residue, a polypeptide of the formula (II)wherein A₁, A₃, A₆ and A₇ are arginine residues, A₂ is a naphthylalanineresidue, A₄ is a lysine residue, X is a D-citrullyl-proline residue, andA₅ is a tyrosine residue, a polypeptide of the formula (II) wherein A₁,A₆ and A₇ are arginine residues, A₂ is a naphthylalanine residue, A₃ isa citrulline residue, A₄ is a lysine residue, X is a D-citrullyl-prolineresidue, A₅ is a tyrosine residue, and a polypeptide of the formula (II)wherein A₁ is a citrulline residue, A₂ is a naphthylalanine residue, A₃,A₆ and A₇ are arginine residues, A₄ is a lysine residue, X is aD-citrullyl-proline residue, A₅ is a tyrosine residue.

The peptides of formula (II) may be exemplified in another embodiment bya peptide of the formula (II) wherein A₁, A₆ and A₇ are arginineresidues, A₂ is a naphthylalanine residue, A₃ is a alanine residue, A₄is a lysine residue, X is a D-lysyl-proline residue, and A₅ is atyrosine residue, a polypeptide of the formula (II) wherein A₁ is acitrulline residue, A₂ is a naphthylalanine residue, A₃, A₆ and A₇ arearginine residues, A₄ is a lysine residue, X is a D-lysyl-prolineresidue, and A₅ is a tyrosine residue, a polypeptide of the formula (II)wherein A₁, A₃ and A₇ are arginine residues, A₂ is a naphthylalanineresidue, A₄ is a lysine residue, X is a D-lysyl-proline residue, A₅ is atyrosine residue, and A₆ is a citrulline residue, a polypeptide of theformula (II) wherein A₁ and A₃ are citrulline residues, A₂ is anaphthylalanine residue, A₄ is a lysine residue, X is a D-lysyl-prolineresidue, A₅ is a tyrosine residue, A₆ and A₇ are arginine residues, anda polypeptide of the formula (II) wherein A₁, A₃ and A₇ are arginineresidues, A₂ is a naphthylalanine residue, A₄ is a lysine residue, X isa D-citrullyl-proline residue, A₅ is a tyrosine residue, and A₆ is acitrulline residue.

The amino acid of A₇ as presented in formula II herein is preferably onein which the carboxyl group is amidated for improving stability of thepolypeptide in vivo such as in serum, etc.

A peptide of the present invention includes a peptide or its amide,ester or salt containing the amino acid sequence which is substantiallythe same amino acid sequence as the sequence of any of theabove-mentioned peptides. Here, “substantially the same amino acidsequence” means an amino acid sequence that is qualitatively identicalin the activity of the peptide or the biological activity of the peptide(e.g. MIP3α secretion) or the like. Accordingly, quantitative variancesare acceptable to some extent (e.g. about 0.01 to 100 times, preferably0.5 to 20 times, or more preferably 0.5 to 2 times). Therefore, one ormore of the amino acids in the amino acid sequences indicated in any ofthe above-mentioned formula (I), (II) and SEQ ID NOS:1-72 can havevariances, so far as they have any of the above-mentioned properties.That is to say, in the present invention, any peptide (variant peptide)resulting from the variance in the amino acid sequence such assubstitution, deletion or insertion (addition) etc. which brings aboutany significant change (i.e. a qualitatively different change, or aqualitatively identical but quantitatively significantly differentchange) in the physiological property or chemical property of theoriginal (non-variant) peptide is deemed as substantially the same asthe original (non-variant) peptide having no such variance, and, theamino acid sequence of such variant peptide is deemed as substantiallythe same as the amino acid sequence of the original (non-variant)peptide.

It is a well-known fact that generally, the changes such assubstitution, deletion or insertion (addition) of an amino acid in apeptide sequence often do not make a significant change to physiologicalproperties or chemical properties of such peptide. For example, it isgenerally considered that substitution of a certain amino acid byanother amino acid of similar chemical properties results in a peptidehaving minimized deviation from the properties of the original peptide.

Amino acids are classified, using the similarity of their properties asto one of the criteria, into the following classes, for example: (i)nonpolar (hydrophobic) amino acids (examples: alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, methionine,etc.); (ii) polar (neutral) amino acids (examples: glycine, serine,threonine, cysteine, tyrosine, asparagine, glutamine, etc.); (iii) basicamino acids carrying positive electric charge (examples: arginine,lysine, histidine, etc.); (iv) acidic amino acids carrying negativeelectric charge (examples: asparatic acid, glutamic acid, etc.), andaccordingly, amino acid substitution within each class can beconservative with regard to the property of a peptide (namely,substitution generating “substantially same” amino acid sequences).

In other words, “substantially the same amino acid sequences” mayinclude:

(i) amino acid sequences wherein 1 or more, or, in other embodiments, 1to 3 amino acids were substituted by other amino acids in the amino acidsequences indicated in the above-mentioned formula (I), (II) and SEQ IDNOS:1-72;

(ii) amino acid sequences wherein 1 or more, or, in other embodiments, 1to 3 amino acids were deleted in the amino acid sequences indicated inthe above-mentioned formula (I), (II) and SEQ ID NOS:1-72;

(iii) amino acid sequences wherein 1 or more or, in other embodiments, 1to 3 amino acids were added (inserted) in the amino acid sequencesindicated in the above-mentioned formula (I), (II) and SEQ ID NOS:1-72;or

(iv) peptides including modifications to amino acids (particularly, theside chains thereof) among the peptides having the amino acid sequencesindicated in above (i), (ii) or (iii), or esters, amides or saltsthereof.

A peptide of the present invention, if and when the substitution,deletion, insertion (addition), modification, etc. of above (i) to (iv)is intentionally or incidentally provided in the amino acid sequencethereof, can be varied to a stable peptide against heat or protease or ahigh-activity peptide having more enhanced activity. The peptides of thepresent invention include also these variant peptides or amides thereof,esters thereof or salts thereof.

Furthermore, among the peptides of the present invention are the peptideconsisting of the amino acid sequence indicated in any of theabove-mentioned formula (I), (II) and SEQ ID NOS:1-72, and the peptidecontaining the amino acid sequence sharing the homology of about 50 to99.9% (preferably, 70 to 99.9%, more preferably 90 to 99.9%) with theforegoing amino acid sequence and having the activities of substantiallythe same nature as the peptide consisting of the amino acid sequenceindicated in any of the above-mentioned formula (I), (II) and SEQ IDNOS:1-72, or amides thereof, esters thereof or salts thereof.

The amides, esters or salts of the peptide having the amino acidsequence indicated in any of the above-mentioned SEQ ID NOS:1-72 includethe same ones as are exemplified for the peptide indicated in theabove-mentioned formula (I). Preferably, the peptide having the aminoacid sequence indicated in any of the above-mentioned SEQ ID NOS:1-72 isamidated at the carboxyl group of the C-terminal amino acid residue.

The peptides of the present invention including the peptide containingthe amino acid sequence indicated in any of the above-mentioned SEQ IDNOS:1-72 can be produced by conventionally known methods of synthesizingpeptides. For the syntheses of peptides, either solid phase peptidesynthesis or liquid phase synthesis may be utilized. Namely, an expectedpeptide can be produced by condensing a partial peptide able toconstitute a peptide or an amino acid with remaining portions, and ifthe product has a protecting group, by eliminating the protecting group.As the known condensation methods and elimination of protecting groups,the following examples (1) to (5) are included:

-   (1) Bodanszky and Ondetti, Peptide Synthesis, Interscience    Publishers, New York (1966).-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York    (1965).-   (3) Izumiya et al., Peptide Synthesis, Basics and Practice, Maruzen,    Tokyo (1975).-   (4) Yajima et al., Protein Chemistry IV, Tokyo Kagakudojin, Tokyo,    pp. 205 (1977).-   (5) Yajima and Zoku-Iyakuhin-no-Kaihatsu, Peptide Synthesis,    Hirokawa Publishing Co., Tokyo, Vol. 14 (1991).    As practical methods for syntheses of peptides, the following    examples can be given:

Generally, commercially available resins for synthesis of polypeptidescan be used. Such resins include, for example, chloromethyl resin,hydroxymethyl resin, benzhydroxylamine resin, aminomethyl resin,4-hydroxybenzylalcohol resin, 4-methylbenzhydroxylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetoamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimetoxyphenyl-hydroxymethyl)phenoxy resin,4-2′,4′-dimetoxyphenyl-Fmoc aminoethylphenoxy resin, etc. Using suchresin, an amino acid with suitably protected α-amino group and sidechain functional group is condensed on the resin to the sequence of theexpected polypeptide in accordance with, conventionally knowncondensation methods. In the last stage of the reaction, the polypeptideis cleared from the resin and simultaneously various protective groupsare removed, and then, by carrying out intramolecular disulfidebond-forming reaction in highly diluted solution, the expectedpolypeptide or amide thereof is obtained. For the above-mentionedcondensation of the protected amino acid, various activated reagentsusable for the syntheses of polypeptides can be used, but it isparticularly better to use carboxyimides. Among such carboxyimides areDCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)cabodiimde, etc. For the activation bythese, together with racemization inhibitory additives (for example,HOBt, HOOBt), a protected amino acid is added directly to the resin, orafter activating the protected amino acid as symmetric acid anhydride orHOBt ester or HOOBt ester, it can be added to ester resin.

Solvents used for the activation of protected amino acids and thecondensation with resins can be chosen from among the solvents known tobe usable for polypeptide condensation reactions. For example, acidamides such as N,N-dimethylformamide, N,N-dimethylacetoamide andN-methylpyrrolidone, halogenated hydrocarbons such as methylene chlorideand chloroform, alcohols such as trifluoroethanol, sulfoxides such asmethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran,nitriles such as acetonitrile and propionitrile, esters such as methylacetate and ethyl acetate, or appropriated mixtures of the foregoing areused. A solvent used for activation of a protected amino acid or itscondensation with resin can be selected from among the solvents known tobe usable for condensing reactions of polypeptides. The reactiontemperature is appropriately set within the scope known to be applicableto polypeptide bond forming reactions, usually, at −20° C. to 50° C.Activated amino acid derivatives are usually used at 1.5 to 4 timesexcess. According to the result of tests adopting ninhydrin reaction, ifthe condensation is insufficient, the repetition of condensationreactions without eliminating protective groups can lead to sufficientcondensation. If sufficient condensation is attained by the repetitionof reactions, unreacted amino acids can be acetylated by the use ofacetic anhydride or acetylimidazole.

The protective group of the amino group used as ingredients include, forexample, Z, Boc, tertialypentyloxycarbony, isobornyloxycarbonyl,4-methoxybenzyloxycabonyl, Cl—Z, Br—Z, adamantyloxycabonyl,trifluoroacetyl, phtaloyl, formyl, 2-nitrophenylsulphenyl,diphenylphosphinothioyl, Fmoc, etc. Carboxyl group can be protected, forexample, by alkyl esterification (e.g. straight-chain, branching orcircular alkyl esterification of methyl, ethyl, propyl, butyl,tertialbutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,2-adamantyl, etc.), aralkyl esterification (e.g. benzylester,4-nitrobenzylester, 4-methoxybenzylester, 4-chlorbenzylester, benzhydrylesterification), phenacylesterification, benzylcarbonylhydrazidation,tertialybutoxycarbonylhydrazidation, tritylhydrazidation, etc. Thehydroxyl group of serine can be protected, for example, byesterification or etherification. The groups suitable for thiseterification include, for example, groups derivatized from carboxylicacid such as lower alkanoyl group such as acetyl group, aroyl group suchas benzoyl group, benzyloxycarbonyl group, ethoxycarbonyl group. Thegroups suitable for etherification include, for example, benzyl group,tetrahydropiranyl group, tertiarybutyl group, etc. As the protectivegroups of phenolic OH group of tyrosine, for example, Bzl, Cl2-Bzl,2-nitrobenzyl, Br—Z, tertiarlybutyl, etc. are used. As the protectivegroups of imidazole of histidine, for example, Tos,4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, Fmoc etc. are used.

Ingredients with activated carboxyl groups include, for example,corresponding acid anhydride, azide, active ester [ester of alcohol(e.g. pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol,cyanomethylalcohol, p-nitrophenol, HONB, N-hydroxysuccimide,N-hydroxyphtalimide, HOBt] are used. Ingredients with activated aminogroup include, for example, corresponding phosphoric amide. As themethods to remove (elimiate) protective groups, for example, catalyticreduction in hydrogen airstream in the presence of a catalyst such asPd-black or Pd-carbon, acid treatment by anhydrous hydrogen fluoride,methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroaceticacid or a mixture thereof, etc., base treatment bydiisopropylethylamine, triethylamine, piperidine, piperadine, etc., andreduction by natrium in liquid ammonia are used. Elimination reaction bythe above-mentioned acid treatment is done generally at the temperatureof about −20° C. to 40° C., but in the acid treatment, it is effectiveto add a cation trapping agent such as anisole, phenol, thioanisole,m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol,1,2-ethanedithiol. 2,4-dinitrophenyl group used as the protective groupof imidazole of histidine is removed by thiophenol treatment. Formylgroup used as the protective group of indole of tryptophan is removed byelimination of protection by the above-mentioned acid treatment in thepresence of 1,2-ethanedithiol, 1,4-butanedithiol, etc. and also isremoved by alkaline treatment by dilute sodium hydroxide solution,dilute ammonia, etc.

Protection and protective group of functional groups not to be involvedin the reaction of ingredients, and elimination of such protectivegroup, and activation of functional groups to be involved in thereaction, etc. can be appropriately selected from among conventionallyknown groups or conventionally known measures. As alternative methods toobtain amides of polypeptides, there is, for example, a method tomanufacture, after amidating and protecting a-carboxyl group ofcarboxy-terminal amino acid and then extending the peptide chain to thedesired chain length on the side of amino group, a polypeptideeliminating the protective group of α-amino group of N-terminal of suchpeptide chain and a polypeptide eliminating the protective group ofcarboxyl group of C-terminal, and then these two peptides are condensedin the above-mentioned mixed solvent. The details of the condensationreaction are the same as described above. After purifying the protectedpolypeptide obtained by the condensation, the desired raw polypeptidecan be obtained by eliminating all the protective groups by theabove-mentioned method. Having purified this raw polypeptide usingvarious known purification methods, if the main fraction isfreeze-dried, an amide type of the desired polypeptide can be obtained.To get an ester type of the polypeptide, for example, make an amino acidester by condensing a-carboxyl group of carboxy-terminal amino acid withthe desired alcohols, and then, the ester type of the desiredpolypeptide can be obtained in the same way as the amide type of thepolypeptide.

After the reaction, the peptides of the present invention can bepurified and isolated by combining usual purification methods such assolvent extraction, distillation, column chromatography, liquidchromatography, re-crystallization, etc. If a peptide obtained by theabove-mentioned methods is a salt-free type, it can be converted to asuitable salt by known methods, or if such peptide is a salt, it can beconverted to a salt-free type by known methods.

Pharmaceutical Compositions and Kits

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier”, which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences”, MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference (Remington: The Science and Practice of Pharmacy, Gennaro, A.,Lippincott, Williams & Wilkins, Philadelphia, Pa., 20^(th) ed, 2000).

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

The pharmaceutical compositions of the invention are suitable foradministration systemically or in a local manner, for example, viainjection of the pharmaceutical composition directly into a tissueregion of a patient (e.g. intralesional injection).

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water-based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters such as ethyl oleate, triglycerides, orliposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe active ingredients, to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., a sterile, pyrogen-free,water-based solution, before use.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum, tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

In yet another aspect, there is provided a pharmaceutical compositioncomprising as active ingredients a peptide having an amino acid sequenceas set forth in SEQ ID NO:1 or an analog or derivative thereof andrapamycin or a derivative thereof.

In another aspect, there is provided a kit comprising i) a peptidehaving an amino acid sequence as set forth in SEQ ID NO:1 or an analogor derivative thereof and ii) a chemotherapeutic agent comprisingrapamycin or a derivative thereof.

In one embodiment, there is provided a pharmaceutical pack containing acourse of anti-neoplastic treatment for one individual mammal comprisinga container having a unit of a T-140 analog of the invention in unitdosage form, and a container having a unit of rapamycin.

In some embodiments, the combinations of the invention are provided inpacks in a form ready for administration. In other embodiments, thecombinations of the invention are provided in concentrated form inpacks, optionally with the diluent required to make final solution(s)for administration. In still other embodiments, the product contains acompound useful in the invention in solid form and, optionally, aseparate container with a suitable solvent or carrier for the compounduseful in the invention.

In still other embodiments, the above packs/kits include othercomponents, e.g., instructions for dilution, mixing and/oradministration of the product, other containers, syringes, needles, etc.Other such pack/kit components will be readily apparent to one of skillin the art.

In a particular embodiment, the kits further comprise instructions foradministering said peptide and said chemotherapeutic agent to a subjectafflicted with cancer, particularly with a tumor of hematopoietic orglial origin, as detailed herein.

Rapamycin, or sirolimus, is an immunosuppressive agent. Sirolimus is amacrocyclic lactone produced by Streptomyces hygroscopicus. The chemicalname of sirolimus is(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.Its molecular formula is C₅₁H₇₉NO₁₃ and its molecular weight is 914.2.The structural formula of sirolimus is shown below:

As defined herein, the term “rapamycin” defines a class ofimmunosuppressive compounds which contain the basic rapamycin nucleus(shown above). The rapamycins of this invention include compounds whichmay be chemically or biologically modified as derivatives of therapamycin nucleus, while still retaining immunosuppressive properties.Accordingly, the term “rapamycin” includes esters, ethers, oximes,hydrazones, and hydroxylamines of rapamycin, as well as rapamycins inwhich functional groups on the rapamycin nucleus have been modified, forexample through reduction or oxidation. The term “rapamycin” alsoincludes pharmaceutically acceptable salts of rapamycins, which arecapable of forming such salts, either by virtue of containing an acidicor basic moiety.

Rapamycin is currently available as RAPAMUNE® (Wyeth-Ayerst) OralSolution and Tablets, indicated for the prophylaxis of organ rejectionin patients receiving renal transplants.

Therapeutic Uses

In another aspect, the invention provides a method for treating asubject afflicted with a tumor selected from the group consisting ofmultiple myeloma and glioma, comprising administering to the subject atherapeutically effective amount of a peptide having an amino acidsequence as set forth in SEQ ID NO:1 or an analog or derivative thereof.

The invention is further directed to a method for inducing hematopoietictumor cell death in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a peptide having anamino acid sequence as set forth in SEQ ID NO:1 or an analog orderivative thereof.

In one particular embodiment, the tumor is multiple myeloma. In anotherparticular embodiment, the tumor is microglioma.

The invention is further directed to a method for inducing glial tumorcell death in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a peptide having an aminoacid sequence as set forth in SEQ ID NO:1 or an analog or derivativethereof.

In one embodiment, the tumor is glioma.

In another aspect, there is provided a method for increasing thesensitivity of tumor cells to an anti-cancer agent in a subject in needthereof comprising administering to the subject a sensitizing-effectiveamount of a peptide having an amino acid sequence as set forth in SEQ IDNO:1 or an analog or derivative thereof in concurrent or sequentialcombination with the anti-cancer agent.

In another embodiment, the anti-cancer agent is a chemotherapeutic drug.Various chemotherapeutic drugs are known in the art, for examplealkylators including, but not limited to, busulfan (Myleran, Busulfex),chlorambucil (Leukeran), ifosfamide (with or without MESNA),cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM(Alkeran), dacarbazine (DTIC-Dome), and temozolamide (Temodar);anthracyclines, including, but not limited to doxorubicin (Adriamycin,Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin),valrubicin (Valstar), and epirubicin (Ellence); antibiotics, including,but not limited to, dactinomycin, actinomycin D (Cosmegen), bleomycin(Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome);aromatase inhibitors, including, but not limited to anastrozole(Arimidex) and letroazole (Femara); bisphosphonates, including, but notlimited to zoledronate (Zometa); cyclo-oxygenase inhibitors, including,but not limited to, celecoxib (Celebrex); estrogen receptor modulatorsincluding, but not limited to tamoxifen (Nolvadex) and fulvestrant(Faslodex); folate antagonists including, but not limited tomethotrexate and tremetrexate; inorganic aresenates including, but notlimited to arsenic trioxide (Trisenox); microtubule inhibitors (e.g.taxanes) including, but not limited to vincristine (Oncovin),vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine(Navelbine), epothilone B or D or a derivative of either, anddiscodermolide or its derivatives, nitrosoureas including, but notlimited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine(BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt); nucleosideanalogs including, but not limited to mercaptopurine, 6-MP (Purinethol),fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine),hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine(FUDR), fludarabine (Fludara), pentostatin (Nipent), cladribine(Leustatin, 2-CdA), gemcitabine (Gemzar), and capecitabine (Xeloda);osteoclast inhibitors including, but not limited to pamidronate(Aredia); platinum containing compounds including, but not limited tocisplatin (Platinol) and carboplatin (Paraplatin); retinoids including,but not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin),and bexarotene (Targretin); topoisomerase 1 inhibitors including, butnot limited to topotecan (Hycamtin) and irinotecan (Camptostar);topoisomerase 2 inhibitors including, but not limited to etoposide,VP-16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate(Etopophos); and tyrosine kinase inhibitors including, but not limitedto imatinib (Gleevec).

For example, the following agents are used in the treatment ofhematopoietic tumors such as lymphomas, and may be used in combinationwith the T-140 analogs of the invention:

DNA-Altering Drugs (Alkylating Agents)

These drugs change DNA, the building block of cells, to prevent cellgrowth, e.g. Bendamustine, Carboplatin (Paraplatin®), Carmustine(BCNU®), Chlorambucil (Leukeran®), Cisplatin (Platinol®),Cyclophosphamide injection (Cytoxan®), Cyclophosphamide oral (Cytoxan®),Dacarbazine (DTIC®), Ifosfamide (ifex®), Lomustine (CCNU®),Mechlorethamine (nitrogen mustard, Mustargen®), Melphalan (Alkeran®) andProcarbazine (Matulane®).

Anti-Tumor Antibiotics

These drugs interact with DNA and decrease cell survival, e.g. Bleomycin(Blenoxane®), Doxorubicin (Adriamycin®, Rubex®), Doxorubicin, Liposomal(Doxil), Idarubicin (Idamycin®) and Mitoxantrone (Novantrone®).

Antimetabolites

These drugs interfere with normal cell growth, e.g. Chlorodeoxyadenosine(Cladribine®, also known as 2-chlorodeoxyadenosine; 2-CdA), CytarabineIV (cytosine arabinoside, Ara-C, Cytosar), Fludarabine IV (Fludara®),Fludarabine oral (Fludara®), Gemcitabine (Gemzar®), Mercaptopurine oral(Purinethol®), Methotrexate oral (Rheumatrex®) Other name: amethopterin,Pentostatin IV (Nipent®) and Thioguanine oral (Lanvis®).

DNA Repair Enzyme Inhibitors

These drugs act on certain proteins (enzymes) that normally work torepair faulty DNA and therefore make cells more likely to die when theyare injured, e.g. Etoposide oral (VP-16, VePesid®, Etopophos) andEtoposide IV (VP-16, VePesid®, Etopophos).

Microtubule Inhibitors

These drugs damage cell structures required for cells to divide, e.g.Vinblastine (Velban®), Vincristine (Oncovin®) and Vinorelbine(Navelbine®).

Steroidal (Corticosteroids)

Corticosteroids, including Prednisone, Prednisolone, Methylprednisoloneand Dexamethasoneare are a group of synthetic hormones closely relatedto cortisol (a glucocorticoid), a natural hormone produced in theadrenal cortex.

Prednisone decreases inflammation by preventing white blood cells fromfunctioning properly. More specifically, the drug interferes withlymphocytes (one of several types of white blood cells). The presence ofwhite blood cells result in inflammation (for many reasons, damage totissue, fungus, virus, bacteria, allergens and almost any foreigninvader)—they go to a site and their presence inflames the area.Prednisone causes lymphocytes to break apart and die.

Examples: Dexamethasone (Decadron®), Methylprednisolone (Medrol®) andPrednisone (Deltasone®).

It should be understood, that the T-140 peptides of the invention may beadministered to augment the anti-cancer effect of a chemotherapeuticdrug, as well as of a combination of drugs. In another particularexample, treating transformed indolent, or aggressive lymphomas thatexpress CD20 may be performed by administering a T-140 analog of theinvention in combination with the following drugs: Cyclophosphamide(Cytoxan®) Doxorubicin (Adriamycin®) Vincristine (Oncovin®) Prednisone(Deltasone®) (collectively known as CHOP chemotherapy) and optionallyRituxan (an anti-CD20 monoclonal antibody).

Suitable dosages and administration routes of chemotherapeutic drugs arereadily determined by the skilled artisan.

In another particular embodiment wherein the drug is rapamycin or aderivative thereof.

In another embodiment, the tumor is a hematopoietic tumor. In aparticular embodiment, said tumor is multiple myeloma. In anotherparticular embodiment, said tumor is microglioma. In another particularembodiment, said tumor is glioma.

In various embodiments of the present invention, the subject is selectedfrom humans and non-human mammals. In a preferable embodiment, thesubject is human.

In another aspect, the invention is directed to the use of apharmaceutical composition comprising a peptide having an amino acidsequence as set forth in SEQ ID NO:1 or an analog or derivative thereofand optionally a chemotherapeutic drug (e.g. rapamycin), for thepreparation of a medicament for treating a subject having a tumorselected from the group consisting of multiple myeloma, microglioma andglioma, for inducing hematopoietic tumor cell death, for inducing glialtumor cell death, and/or for increasing the sensitivity of tumor cellsto an anti-cancer agent.

In another aspect, the invention is directed to a pharmaceuticalcomposition comprising a peptide having an amino acid sequence as setforth in SEQ ID NO:1 or an analog or derivative thereof and optionally achemotherapeutic drug (e.g. rapamycin) for treating a subject having atumor selected from the group consisting of multiple myeloma,microglioma and glioma, for inducing hematopoietic tumor cell death, forinducing glial tumor cell death, and/or for increasing the sensitivityof tumor cells to an anti-cancer agent.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans. An exemplary dosage range for human use may be from about 0.05to about 10 mg/kg per administration (e.g. subcutaneously, once or twicea day).

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES

Reagents

AMD3100 was purchased from Sigma, Israel. 4F-benzoyl-TN14003 (SEQ IDNO:1) was synthesized by Novotide Ltd. In order to neutralize theactivity of 4F-benzoyl-TN14003 in some experiments proteinase K was usedas followed: 4F-benzoyl-TN14003 was incubated with Proteinase K (Dako, 1mg/ml) for 20 min at 37° C. following by 10 min incubation at 95° C.

The label “4FB-TN14003” as it appears throughout the figures, is used toindicate 4F-benzoyl-TN14003 (SEQ ID NO:1).

Example 1 4F-Benzoyl-TN14003 Induces MIP3α Secretion from Prostate CellLines in a CXCR4 Agonist Manner

A PC3 prostate cell line that overexpresses high levels of CXCR4 wasgenerated. Single cell clones were generated from this PC3-CXCR4 cellline, and one of the clones (PC3-CXCR4.5), which showed a high andstable expression level of CXCR4, was selected for the experiments. FIG.1A presents FACS histograms of PC3 cells (left panel) and a single cellclone with stable overexpression of CXCR4, GFP and luc genes(PC3—CXCR4.5, right panel) that were stained for the control (IgG2a-PE,full histograms) and CXCR4 (IgG2a-12G5, empty histograms) antibodies.

In this example, regulation of the chemokine MIP3α (macrophageinflammatory protein 3α) was examined. It was found that PC3-CXCR4.5cells secreted higher levels of MIP3α than PC3 cells, and increasingdoses of CXCL12 increased the secretion of MIP3α in both PC3 andPC3-CXCR4.5 cells (FIG. 1B). In FIG. 1B, PC3 (left panel) andPC3—CXCR4.5 (right panel) cells were stimulated with the indicatedconcentrations of CXCL12 for 48 hours and MIP3α secretion was assessedby ELISA. The results represent the average of triplicates±STDEV.

In PC3 cells, treatment with neutralizing antibodies against CXCL12(αCXCL12) or with Pertussis toxin (PTX; alone or in combination withCXCL12) effectively inhibited the secretion of MIP3α (FIG. 1C). Incontrast, in PC3-CXCR4.5 cells, anti-CXCL12 antibodies did not quiteaffect the secretion level of MIP3α; with PTX treatment alone, secretionof MIP3α was decreased, but PTX in combination with CXCL12 did notdemonstrate an inhibitory effect on the level of MIP3α (FIG. 1C). Theeffects of the CXCR4 antagonist AMD3100 and of 4F-benzoyl-TN14003(4FB-TN14003), hitherto known as a CXCR4 antagonist, on MIP3α secretionwere further tested. Surprisingly, 4F-benzoyl-TN14003, but not AMb3100induced in both cell lines MIP3α secretion in an agonist manner (FIG.1C). The effect of 4F-benzoyl-TN14003 was partially inhibited by PTXtreatment in both cell lines (FIG. 1C). In FIG. 1C, CXCR4 signaling inPC3 (left panel) and PC3-CXCR4.5 (right panel) cells was inhibited withanti-CXCL12 antibodies (αCXCL12) and Pertussis toxin (PTX) treatmentsalone or in combination with CXCL12, as indicated. Secretion of MIP3αwas assessed by ELISA. The results represent the average oftriplicates±STDEV.

PC3-CXCR4.5 cells secreted higher levels of MIP3α than PC3 cells, andincreasing doses of CXCL12 increased the secretion of MIP3α from thesecells. Spontaneous secretion of MIP3α is CXCL12 and PTX independent inthese cells. The spontaneous, CXCL12-induced and4F-benzoyl-TN14003-induced secretion was found to be CXCR4 dependent, asdetermined by evaluation of CXCR4 and MIP3α expression followingtransfection with CXCR4-specific or control siRNA.

Example 2 The Effect of 4F-Benzoyl-TN14003 and AMD-3100 on Cell Survivaland Proliferation

The effect of CXCR4 antagonists on the survival of freshly isolatednormal CD4⁺ T cells, CD34⁺ stem cells and keratinocytes was examined.The proliferation and survival of keratinocytes was not inhibited byCXCR4 antagonists AMD-3100 (20 μM) and 4F-benzoyl-TN14003 (4-20 μM) orthe natural ligand CXCL12 (FIG. 2C). The proliferation and survival ofCD4⁺ T cells was not inhibited by CXCR4 antagonists AMD-3100 (20 μM).However, both 4F-benzoyl-TN14003 (4-20 μM) and CXCL12, induced T cellproliferation (FIG. 2A). The proliferation and survival of CD34⁺ stemcells was partially inhibited (−25%) by 4F-benzoyl-TN14003 (8, 20 μM),whereas, CXCL12 and AMD-3100 (20 μM) did not affect cell numbers (FIG.2B). in FIG. 1, the results represent average of triplicates±STDEV.

Next, the effect of the CXCR4 modulators on PC3 and PC3-CXCR4.5 prostatetumor cells was tested. Similarly to CXCL12, both AMD-3100 and4F-benzoyl-TN14003 induced the proliferation of prostate cancer cells(FIG. 3A). The proliferation induced by 4F-benzoyl-TN14003 wasdose-dependent (FIG. 3B). Other cell lines such as breast carcinoma,SKBR3, prostate carcinoma, 22Rv1, and glioblastoma, U-87, were eitherstimulated or not affected by CXCL12 and CXCR4 modulators AMD-3100 and4F-benzoyl-TN14003 (FIGS. 4A-4C, respectively). These data suggest that4F-benzoyl-TN14003 can increase proliferation of prostate cells PC3, andPC3-CXCR4.5 cells and can act as an agonist.

Overall, these results indicate that 4F-benzoyl-TN14003 does not induceepithelial tumor cell death and can in some cases stimulate growth ofepithelial cells expressing CXCR4. CXCR4 is expressed on a majority ofhematopoietic stem cells. Therefore the effect of CXCR4 antagonists onthe survival and proliferation of a variety of blood borne hematopoietictumor cell lines was tested.

The proliferation and survival of the CML K562 cells or K562 cells thatover-express CXCR4 (indicated “CXCR4 high” or “K562-CXCR4”) was firststudied. It was found that the CXCR4 modulator 4F-benzoyl-TN14003 (4-20μM), but not AMD-3100, inhibited the growth and stimulated apoptoticcell death (measured by Annexin V assay in accordance with themanufacturer's recommendation; IQP, Groningen, Netherlands) of K562cells that express low and high levels of CXCR4 (FIG. 5).

The inhibitory effect of 4F-benzoyl-TN14003 was further studied usingthe promyelocytic leukemic cell line HL-60 and the AML cell line NB4.The CXCR4 modulator 4F-benzoyl-TN14003 (4-20 μM), but not AMD-3100, wasfound to inhibit the growth and stimulate apoptotic cell death (measuredby Annexin V assay) of HL-60 cells (FIGS. 6A and 6B, respectively).Pretreatment of HL-60 cells with AMD-3100 (50 μM) abolished the effectof 4F-benzoyl-TN14003 on these cells (FIGS. 6A and 6C depict tworepresentative experiments). NB4 cells were less sensitive to4F-benzoyl-TN14003 however; the inhibitory effect of 4F-benzoyl-TN14003on NB4 cells was abolished by pretreatment with AMD-3100 (FIG. 6D).

The inhibitory effect of 4F-benzoyl-TN14003 was further studied usingmultiple myeloma (MM) cells. The inventors found that MM cells are themost affected human blood cancer tested. The CXCR4 antagonist4F-benzoyl-TN14003 (0.4-40 μM), but not AMD-3100 or CXCL12, inhibitedthe growth and stimulated apoptotic cell death (measured by annexinassay) of RPMI8226, ARH77, U266, and NCI human MM cells (FIGS. 7A, 7B,7C, 7D, 7E and 7F, respectively).

To verify that the effect of 4F-benzoyl-TN14003 is due to the intactpeptide, the peptide was incubated with proteinease K and the effect oftreated peptide on the survival of human RPMI8226 MM cells (FIGS. 8A and8B) and the migration of Human Jurkat cells in response to CXCL12 (50ng/ml, black columns; FIG. 8C) was tested. As can be seen in FIG. 8,treatment of 4F-benzoyl-TN14003 with proteinease K abolished theactivity of the peptide.

To further test the effect of 4F-benzoyl-TN14003 on the proliferationand survival of RPMI8226MM cells, the cells were incubated in 10% FCSfor 72 hr with different concentrations of 4F-benzoyl-TN14003. Underthese conditions, 4F-benzoyl-TN14003 demonstrated a rapid and sustainedanti-proliferative (FIG. 9A) death-induced (FIGS. 9B-9E 1-72 hr) effecton RPMI8226MM cells. In FIG. 9A, diamonds indicate control cells(incubated without 4F-benzoyl-TN14003); squares indicate cells incubatedwith 8 μM of the peptide; triangles indicate cells incubated with 20 μMof the peptide; circles indicate cells incubated with 50 μM of thepeptide; and crosses indicate cells incubated with 100 μM of thepeptide.

The effect of 4F-benzoyl-TN14003 on the proliferation and survival ofRPMI8226MM cells may be mediated through induction of apoptotic celldeath. Staining of RPMI8226 cells with 7AAD and annexin shows a clearincrease in the population of cells that are annexin⁺7AAD⁻ (FIG. 10C)early apoptotic cells as well as annexin⁺7AAD⁺ late apoptotic deathcells (FIG. 10C). FIG. 10A illustrates that live cells appear at thebottom left region of the plot, necrotic cells appear at the top leftregion, late apoptotic cells appear at the top right region and earlyapoptotic cells appear at the bottom right region.

Example 3 4F-Benzoyl-TN14003 Selectively and Rapidly StimulatesBM-Derived MM Cell Death

Based on the previous results, the effect of 4F-benzoyl-TN14003 on theproliferation and survival of freshly isolated bone marrow cells ofpatients with multiple myeloma (MM) was studied. Bone marrow (BM)derived samples that have high percentage of MM were sensitive totreatment with 4F-benzoyl-TN14003 (FIG. 11A-11C); however, normal BMsamples or BM samples with low percentage of MM cells were not sensitiveto treatment with 4F-benzoyl-TN14003 (FIG. 11D).

Incubation of BM cells from MM patients with 8 μM of 4F-benzoyl-TN14003for 24 hr induced apoptosis of CD138⁺ MM cells (FIG. 12A, R3) whereasthe percentage of CD34⁺ cells in these samples remained unaffected (FIG.12B, R2).

Example 4 4F-benzoyl-TN14003 Synergized with Rapamycin to Induce MM andGlioma Cell Death

One candidate target molecule for anti-tumor therapy is represented bythe phosphoprotein mammalian target of rapamycin, mTOR (also known asFRAP [FKBP12-rapamycin-associated protein], RAFT [rapamycin and FKBP-12target], or RAPT), in which the PI3-K/Akt pathway has been suggested toaffect the mTOR phosphorylation state and catalytic activity. Rapamycinbinds to its cellular receptor, the immunophilin FK506 binding protein12 (FKBP12), to form a complex that interacts with mTOR, therebyblocking its activity. Mitogen-activated signaling through mTORphosphorylates the serine/threonine kinase p70S6K and the translationalrepressor eukaryotic initiation factor (eIF) 4E binding protein (4EBP1)also known as PHAS-I. Activated p70S6K directly phosphorylates the 40Sribosomal protein S6, which correlates with enhanced translation oftranscripts with 5-terminal oligo-pyrimidine sequences that encodecomponents of the translational machinery. Multi-site phosphorylation of4EBP1 results in its dissociation from eIF4E, thereby allowing eIF4E toparticipate in assembly of a translational initiation complex leading totranslational up-regulation of proteins required for cell cycleprogression from G1 to S phase. Currently, rapamycin derivatives such asCCI-779, are in clinical testing for MM (Phase II). Recently, it wasshown that rapamycin can sensitize MM cell lines as well as primary MMcells to dexamethasone-induced apoptosis, an effect being associatedwith a concomitant down-regulation of cyclin D2 and the keyanti-apoptotic protein survivin.

Rapamycin at concentration of 10-100 μM can induce cell cycle arrest ofRPMI8226MM cells. However, it can poorly induce apoptotic cell death ofthese cells (FIG. 13).

Herein, it was tested whether rapamycin can sensitize MM cell lines to4F-benzoyl-TN14003-induced apoptosis. In a concentration of 50 rapamycin(“Rapa”) stimulated 4.8% cell death over control (22.3% compared to17.5%). In contrast to rapamycin, 4F-benzoyl-TN14003 induced 28.9% celldeath over control. When both agents were used in combination, theysynergized to induce 48.9% cell death over control (FIG. 14).

The ability of 4F-benzoyl-TN14003 to induce tumor cell death was alsodemonstrated for glioma cells. U87 cells were treated with4F-benzoyl-TN14003 and tested for their survival (FIG. 15A) and death(FIG. 15B). The results indicate that 4F-benzoyl-TN14003 synergizes withrapamycin to induce apoptosis in U87 cells (measured by an Annexin Vassay).

Example 5 Animal Pharmacology Studies—4F-Benzoyl-TN14003 Demonstrates anIn Vivo Anti-Tumor Effect Against Human NB4 Leukemic Cells in aXenograft Model

In a number of in-vivo studies, 4F-benzoyl-TN14003 was found todemonstrate in vivo anti-tumor effects against human NB4 and K562leukemic cells in a xenograft model as detailed herein.

Acute promyelocytic leukemia (APL)-derived NB4 cell is extensively usedas an in vitro and in vivo model system for studying human leukemia. Toinvestigate the therapeutic anti-tumor potential of the CXCR4 antagonist4F-benzoyl-TN14003 against human leukemic cells, 10×10⁶ NB4 cells wereinjected subcutaneously (s.c) into immunodeficient nude mice (n=5).After 14 days, tumors were established at the size of 0.4 cm², and micewere divided into 4F-benzoyl-TN14003-treated (8 mg/Kg, open squares) andPBS-treated (control, full diamonds) groups. The 4F-benzoyl-TN14003 drugsolution was administered intraperitoneal (i.p) each day for 5 days,followed by 2 days without drug, and then 4 additional daily injections(total of 9 injections, FIG. 16). As can be seen in FIG. 16, in micethat were treated with 4F-benzoyl-TN14003, tumor growth was arrested.

To further test the effect of 4F-benzoyl-TN14003 against human leukemiccells, 5×10⁶ NB4 cells were injected subcutaneously (s.c) intoimmunodeficient nude mice (n=5). After 14 days, tumors were very small,at the size of 0.02 cm², and mice were divided into4F-benzoyl-TN14003-treated (open squares) and PBS-treated (control, fulldiamonds) groups. The 4F-benzoyl-TN14003 drug solution was administeredsubcutaneously (8 mg/Kg) each day for 4 days (total of 4 injections,FIG. 17A). On day 20, all control mice developed tumors (0.49±0.1 cm²),whereas in the treated mouse group, only 1 mouse developed a smalltumor.

On day 31, all control mice developed big tumors in size (FIG. 17A) andweight (FIG. 17B), whereas in the treated group, 4/5 mice developedsmaller tumors in size and weight.

These results suggest potential therapeutic anti-leukemic use of4F-benzoyl-TN 14003.

Example 6 4F-Benzoyl-TN14003 Demonstrates an In Vivo Anti-Tumor EffectAgainst Human K562 Leukemic Cells in a Xenograft Model

To further investigate the use of 4F-benzoyl-TN14003 in CML tumordevelopment in vivo, an animal model for CML was established in NOD/SCIDmice. The NOD/SCID mice model is a powerful predictor of the clinicalcourse of leukemia and can provide a good model system to explore thefeasibility of different therapeutic programs. This model enablesmonitoring the progression and localization of leukemic cells in vivo sothat a small number of tumor cells could be detected and the patterns ofgrowth and distribution be studied over time. Current animal models ofhuman leukemic diseases require injection of a large number of targetcells. In addition to that, monitoring therapeutic drugs (i.e. STI571)over time on the same animal has not been possible and it usuallyrequires the unnecessary sacrifice of a large number of animals for eachexperiment. Monitoring leukemic tumor growth, metastases, and responseto therapeutic interventions in animal models of minimal disease statesis critical for the development of effective strategies that targetsmall numbers of leukemic cells, thus avoiding minimal residual disease.

Herein, a novel in vivo model system is presented, which is noninvasive,direct, and facilitates sensitive quantification of the distribution ofleukemic cells in a live animal. In order to monitor the progression andlocalization of leukemic cells in vivo, the human CML tumor cell linesK562 (K562L) was stably transduced with a retrovirus carrying theluciferase gene. This cell line was injected IP into NOD/SCID mice(2.5×10⁵, 5×10⁵, 10⁶, 2×10⁶ per mouse; FIGS. 18 A-D, respectively) thatwere pretreated with a sub-lethal dose of radiation in order to diminishany natural killer cell activity and enhance acceptance of the tumorcells. Twenty-four hr after injection of the cells, the light generatedinternally by the luciferase expressed in the tumor cells and thentransmitted through the animal's tissues was monitored externally usinga cooled charge-coupled device camera (CCCD). The substrate luciferinwas provided exogenously via IP injection. As few as 2.5×10⁵ leukemictumor cells distributed throughout the peritoneal cavity were detectableexternally (FIG. 18A). Quantitation was achieved by integrating thesignal intensity using the Metaview software. The integrated lightdetected from the animals was proportional to the number of cellsinjected IP (FIG. 18).

Two therapeutic approaches were studied using this model. The drugtherapy for CML, STI571 (Gleevec® provided by Novartis), a syntheticpyrimidine derivative, which inhibits selectively the tyrosine-kinaseactivity of c-abl and BCR-ABL, was explored in this model system. Inaddition, the effect of 4F-benzoyl-TN14003 on the growth of cells invivo was investigated. As demonstrated in FIG. 19, STI571 at 40 mg/Kg(diamonds) stimulated a significant reduction in signal intensities inthe treated groups over time.

Further, the effect of 4F-benzoyl-TN14003 on K562L tumor development wasstudied. 4F-benzoyl-TN14003 was injected i.p into NOD/SCID mice in theamount of 4 mg/Kg per mouse/per injection. The first injection of4F-benzoyl-TN14003 was given immediately after the injection of K562-luccells in an opposite route of injection on the peritoneum. Furtherinjections of 4F-benzoyl-TN14003 were given every three days. Mice weremonitored under the CCCD camera, 24 hr after each injection at serialtime points, every three days. As can be seen in FIG. 20, a 50%inhibition in K562L tumor development over time was detected.

To further support the potential use of 4F-benzoyl-TN14003 in treatingK562 CM cells in vivo, an additional experiment comparing its activityto the CXCR4 inhibitor AMD-3100 was performed. 4F-benzoyl-TN14003 wasinjected i.p into NOD/SCID mice in the amount of 2 mg/Kg whereasAMD-3100 was injected 4 mg/Kg per mouse/per injection. The firstinjection was given 2 days after the injection of K562-luc cells.Further injections were given on days 5, 7 and 11. Mice (n=3) weremonitored under the CCCD camera on day 17 after injection of cells. Ascan be seen in FIG. 21, a >90% inhibition in K562L tumor developmentover time was measured upon 4F-benzoyl-TN14003 treatment, but not whenAMD-3100 was administered.

Example 7 4F-Benzoyl-TN14003 Demonstrates In Vivo Anti-Tumor EffectAgainst Human of RPMI8226 Cells MM Cells in a Xenograft Model

RPMI8226 MM cells are used as an in vitro and in vivo model system forstudying human MM. To investigate the therapeutic anti-tumor potentialof 4F-benzoyl-TN14003 against human MM cells, 5×10⁶ NB4 cells wereinjected subcutaneously (s.c) into immunodeficient SCID/Bz mice (n=8 foreach group, p<0.05). Tumors were treated S.C. with two concentration of4F-benzoyl-TN14003 (100 or 300 microgram/mouse) for 21 days. The4F-benzoyl-TN14003 drug solution was administered SC each day for 21days, followed by 14 days without drug. After 35 days, tumors in thecontrol group were established at the size of 0.7 cm², and in thetreated mice tumors were reduced (FIG. 22A). In FIG. 22A, diamondsrepresent RPMI8226 tumors from untreated mice, squares representRPMI8226 tumors from mice treated with 100 μg of 4F-benzoyl-TN14003, andcrosses represent RPMI8226 tumors from mice treated with 300 μg of4F-benzoyl-TN14003.

To further test the effect of 4F-benzoyl-TN14003 on human MM tumors invivo, established tumors having a similar size were treated for 7 dayswith 300 microgram/mouse of 4F-benzoyl-TN14003. As can be seen in FIG.22B, treatment with 4F-benzoyl-TN14003 stimulated the reduction of thetumor size (n=1). In FIG. 22B, diamonds represent tumors from untreatedmice, and squares represent RPMI8226 tumors from mice treated with4F-benzoyl-TN14003.

The effect of 4F-benzoyl-TN 14003 on tumor cells in vivo was furthertested by injection of 4F-benzoyl-TN14003 (300 microgram/mouse) and 24hr later collecting the tumors and staining for apoptosis using theTUNEL assy. Clear massive apoptosis induced by 4F-benzoyl-TN14003 isseen in FIG. 23. In FIG. 23, top left and top right panels,respectively, depict DAPI staining or TUNEL staining of a positivecontrol sample (treated with DNAse I); middle left and middle rightpanels, respectively, depict DAPI staining or TUNEL staining of a sampleobtained from 4F-benzoyl-TN14003 treated mice; and bottom left andbottom right panels, respectively, depict DAPI staining or TUNELstaining of a sample obtained from control mice (not treated with4F-benzoyl-TN14003).

REFERENCES

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The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1. A method for treating a subject having a tumor, comprisingadministering to the subject a therapeutically effective amount of ananti-CD20 antibody and a peptide having an amino acid sequence as setforth in SEQ ID NO:1 or an analog or derivative thereof, therebytreating the subject having the tumor.
 2. The method of claim 1, whereinsaid anti-CD20 antibody comprises Rituxan.
 3. The method of claim 1,wherein the analog or derivative has an amino acid sequence as set forthin formula (I) or a salt thereof: (I)1  2 3   4   5  6 7  8  9 10 11 12  13 14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein: A₁ is an arginine, lysine, ornithine, citrulline, alanine orglutamic acid residue or a N-α-substituted derivative of these aminoacids, or A₁ is absent; A₂ represents an arginine or glutamic acidresidue if A₁ is present, or A₂ represents an arginine or glutamic acidresidue or a N-α-substituted derivative of these amino acids if A₁ isabsent; A₃ represents an aromatic amino acid residue; A₄, A₅ and A₉ eachindependently represents an arginine, lysine, ornithine, citrulline,alanine or glutamic acid residue; A₆ represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue; A₇ represents a proline, glycine, ornithine, lysine, alanine,citrulline or arginine residue; A₈ represents a tyrosine, phenylalanine,alanine, naphthylalanine, citrulline or glutamic acid residue; A₁₀represents a citrulline, glutamic acid, arginine or lysine residue; A₁₁represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized; and the cysteineresidue of the 4-position or the 13-position can form a disulfide bond,and the amino acids can be of either L or D form.
 4. The method of claim1, wherein the peptide is selected from the group consisting of SEQ IDNOS:1-72.
 5. The method of claim 1, wherein the tumor is selected fromleukemia, lymphoma, microglioma and multiple myeloma.
 6. A method forinducing tumor cell death in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-CD20 antibody and a peptide having an amino acid sequence as setforth in SEQ ID NO:1 or an analog or derivative thereof, therebyinducing tumor cell death.
 7. The method of claim 6, wherein saidanti-CD20 antibody comprises Rituxan.
 8. The method of claim 6, whereinthe analog or derivative has an amino acid sequence as set forth informula (I) or a salt thereof: (I)1  2 3   4   5  6 7  8  9 10 11 12  13 14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein: A₁ is an arginine, lysine, ornithine, citrulline, alanine orglutamic acid residue or a N-α-substituted derivative of these aminoacids, or A₁ is absent; A₂ represents an arginine or glutamic acidresidue if A₁ is present, or A₂ represents an arginine or glutamic acidresidue or a N-α-substituted derivative of these amino acids if A₁ isabsent; A₃ represents an aromatic amino acid residue; A₄, A₅ and A₉ eachindependently represents an arginine, lysine, ornithine, citrulline,alanine or glutamic acid residue; A₆ represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue; A₇ represents a proline, glycine, ornithine, lysine, alanine,citrulline or arginine residue; A₈ represents a tyrosine, phenylalanine,alanine, naphthylalanine, citrulline or glutamic acid residue; A₁₀represents a citrulline, glutamic acid, arginine or lysine residue; A₁₁represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized; and the cysteineresidue of the 4-position or the 13-position can form a disulfide bond,and the amino acids can be of either L or D form.
 9. The method of claim6, wherein the peptide is selected from the group consisting of SEQ IDNOS:1-72.
 10. The method of claim 6, wherein the tumor is selected fromleukemia, lymphoma, microglioma and multiple myeloma.
 11. A method forincreasing the sensitivity of tumor cells to an anti-CD20 antibody,comprising administering to a subject in need thereof asensitizing-effective amount of a peptide having an amino acid sequenceas set forth in SEQ ID NO:1 or an analog or derivative thereof inconcurrent or sequential combination with the anti-CD20 antibody,thereby increasing the sensitivity of tumor cells to the anti-CD20antibody.
 12. The method of claim 11, wherein said anti-CD20 antibodycomprises Rituxan.
 13. The method of claim 11, wherein the analog orderivative has an amino acid sequence as set forth in formula (I) or asalt thereof: (I) 1  2 3   4   5  6 7  8  9 10 11 12 13  14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein: A₁ is an arginine, lysine, ornithine, citrulline, alanine orglutamic acid residue or a N-α-substituted derivative of these aminoacids, or A₁ is absent; A₂ represents an arginine or glutamic acidresidue if A₁ is present, or A₂ represents an arginine or glutamic acidresidue or a N-α-substituted derivative of these amino acids if A₁ isabsent; A₃ represents an aromatic amino acid residue; A₄, A₅ and A₉ eachindependently represents an arginine, lysine, ornithine, citrulline,alanine or glutamic acid residue; A₆ represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue; A₇ represents a proline, glycine, ornithine, lysine, alanine,citrulline or arginine residue; A₈ represents a tyrosine, phenylalanine,alanine, naphthylalanine, citrulline or glutamic acid residue; A₁₀represents a citrulline, glutamic acid, arginine or lysine residue; A₁₁represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized; and the cysteineresidue of the 4-position or the 13-position can form a disulfide bond,and the amino acids can be of either L or D form.
 14. The method ofclaim 11, wherein the peptide is selected from the group consisting ofSEQ ID NOS:1-72.
 15. The method of claim 11, wherein the tumor isselected from leukemia, lymphoma, microglioma and multiple myeloma. 16.A pharmaceutical composition comprising as active ingredients (i) apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof and (ii) an anti-CD20 antibody.
 17. Thecomposition of claim 16, wherein the analog or derivative has an aminoacid sequence as set forth in formula (I) or a salt thereof: in formula(I) or a salt thereof: (I) 1  2 3   4   5  6 7  8  9 10 11 12 13  14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein: A₁ is an arginine, lysine, ornithine, citrulline, alanine orglutamic acid residue or a N-α-substituted derivative of these aminoacids, or A₁ is absent; A₂ represents an arginine or glutamic acidresidue if A₁ is present, or A₂ represents an arginine or glutamic acidresidue or a N-α-substituted derivative of these amino acids if A₁ isabsent; A₃ represents an aromatic amino acid residue; A₄, A₅ and A₉ eachindependently represents an arginine, lysine, ornithine, citrulline,alanine or glutamic acid residue; A₆ represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue; A₇ represents a proline, glycine, ornithine, lysine, alanine,citrulline or arginine residue; A₈ represents a tyrosine, phenylalanine,alanine, naphthylalanine, citrulline or glutamic acid residue; A₁₀represents a citrulline, glutamic acid, arginine or lysine residue; A₁₁represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized; and the cysteineresidue of the 4-position or the 13-position can form a disulfide bond,and the amino acids can be of either L or D form.
 18. The composition ofclaim 16, wherein the peptide is selected from the group consisting ofSEQ ID NOS:1-72.
 19. The composition of claim 16, wherein said anti-CD20antibody comprises Rituxan.
 20. A kit containing i) a peptide having anamino acid sequence as set forth in SEQ ID NO:1 or an analog orderivative thereof and ii) (ii) an anti-CD20 antibody.
 21. The kit ofclaim 20, wherein the analog or derivative has an amino acid sequence asset forth in formula (I) or a salt thereof: in formula (I) or a saltthereof: (I) 1  2 3   4   5  6 7  8  9 10 11 12 13  14A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein: A₁ is an arginine, lysine, ornithine, citrulline, alanine orglutamic acid residue or a N-α-substituted derivative of these aminoacids, or A₁ is absent; A₂ represents an arginine or glutamic acidresidue if A₁ is present, or A₂ represents an arginine or glutamic acidresidue or a N-α-substituted derivative of these amino acids if A₁ isabsent; A₃ represents an aromatic amino acid residue; A₄, A₅ and A₉ eachindependently represents an arginine, lysine, ornithine, citrulline,alanine or glutamic acid residue; A₆ represents a proline, glycine,ornithine, lysine, alanine, citrulline, arginine or glutamic acidresidue; A₇ represents a proline, glycine, ornithine, lysine, alanine,citrulline or arginine residue; A₈ represents a tyrosine, phenylalanine,alanine, naphthylalanine, citrulline or glutamic acid residue; A₁₀represents a citrulline, glutamic acid, arginine or lysine residue; A₁₁represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized; and the cysteineresidue of the 4-position or the 13-position can form a disulfide bond,and the amino acids can be of either L or D form.
 22. The kit of claim20, wherein the peptide is selected from the group consisting of SEQ IDNOS:1-72.
 23. The kit of claim 20, wherein said anti-CD20 antibodycomprises Rituxan.